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The function of CCN family proteins is determined by their interactions with multiple cofactors that are present in the microenvironment. Therefore, determining these cofactors is critically important in understanding the molecular function of CCN family members. For this objective, a bacteriophage random peptide display library is a suitable tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of 7-20 random amino acid residues on its surface. Bacteriophage clones that possess peptides that bind to a CCN family protein are selected through several cycles of a process called biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, the oligopeptides that specifically bind to the CCN family member can be specified. The obtained peptide sequences can be utilized to design peptide aptamers for CCN family proteins, or as a key sequence to determine new CCN family cofactor candidates in silico. Instead of a random peptide cDNA library, an antibody cDNA library from naïve lymphocytes or from B cells immunized by a CCN family protein can be used in order to obtain a highly specific CCN family detection or functional modulation tool.

DOI： 10.1007/978-1-0716-2744-0_8

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The method of labeling proteins of interest with fluorescent dyes that can specifically stain organelles in living cells provides a tool for investigating various cellular processes under a microscope. Visualization (imaging) of the cells using fluorescence has many advantages, including the ability to stain multiple cell organelles and intracellular proteins simultaneously and discriminately, and is used in many research fields. In this chapter, we describe the observation of cell organelles using fluorescence staining to analyze the functions of CCN family proteins involved in various cellular events.

DOI： 10.1007/978-1-0716-2744-0_3

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Dental pulp cells (DPCs) differentiate into odontoblasts. To observe odontoblastic differentiation, the detection of dentinogenesis-specific molecules such as dentin sialophosphoprotein (DSPP) and the measurement of alkaline phosphatase (ALP) activity are reliable approaches. CCN family member 2 (CCN2) has been proposed as a marker for dentinogenesis. Our recent study revealed that the expression levels of Ccn4, Ccn5, and Ccn6 were changed in accordance with odontoblastic differentiation. Therefore, Ccn4, Ccn5, and Ccn6, as well as Ccn2, could serve as a comprehensive set of markers for dentinogenesis. Here, we describe a method of measuring the Ccns expression levels in differentiating rat DPCs.

DOI： 10.1007/978-1-0716-2744-0_19

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Although two-dimensional (2D) cultures from bone lineage cells are often used, it is well-known that this culture system is completely different from the in vivo bone matrix environment. In this paper, we describe a 3D culture method using both the mouse osteocytic cell line, MLO-Y4, and an osteocyte-enriched population of the cells isolated from mice. These cells are embedded in collagen gel with recombinant cellular communication network (CCN) factor proteins; then, osteoblasts or osteoclasts are inoculated and cultured on the collagen gel. Because this method mimics the in vitro bone matrix environment, it is useful for understanding the detailed mechanism of actions of CCN proteins in the bone matrix.

DOI： 10.1007/978-1-0716-2744-0_17

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Classically, scientific research has been driven by hypotheses based on personal inspiration and intuition against the background of personal knowledge. In contrast, scientists have recently proposed that scientific research should basically be driven by data, meaning big data yielded by preliminary omic analyses in this context. A genuine hypothesis-driven strategy is usually exciting but occasionally ends up with negative conclusions, whereas a data-driven approach is less exciting and cost-consuming but produces significant outcomes in most cases. Here, we should be aware that a number of bioscientific resources provide a variety of big data free of charge. Therefore, one of the most effective research strategies is to construct a research question based on comprehensive knowledge derived not only from inside information, but also from the analysis of data available to everybody. However, a classical scientist without a sufficient bioinformatic background may hesitate in dealing with information supplied through the Internet. This chapter is aimed at CCN family researchers who do not possess specific bioinformatic knowledge and/or huge grants-in-aid, in order to assist them in developing their research by taking advantage of the scientific treasury open to the public.

DOI： 10.1007/978-1-0716-2744-0_12

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Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1β than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1β in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA.

DOI： 10.3390/ijms232315311

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Fibroblast growth factors (FGFs) constitute a large family of signaling molecules that act in an autocrine/paracrine, endocrine, or intracrine manner, whereas the cellular communication network factors (CCN) family is composed of six members that manipulate extracellular signaling networks. FGFs and CCNs are structurally and functionally distinct, except for the common characteristics as matricellular proteins. Both play significant roles in the development of a variety of tissues and organs, including the skeletal system. In vertebrates, most of the skeletal parts are formed and grow through a process designated endochondral ossification, in which chondrocytes play the central role. The growth plate cartilage is the place where endochondral ossification occurs, and articular cartilage is left to support the locomotive function of joints. Several FGFs, including FGF-2, one of the founding members of this family, and all of the CCNs represented by CCN2, which is required for proper skeletal development, can be found therein. Research over a decade has revealed direct binding of CCN2 to FGFs and FGF receptors (FGFRs), which occasionally affect the biological outcome via FGF signaling. Moreover, a recent study uncovered an integrated regulation of FGF and CCN genes by FGF signaling. In this review, after a brief introduction of these two families, molecular and genetic interactions between CCN and FGF family members in cartilage, and their biological effects, are summarized. The molecular interplay represents the mutual involvement of the other in their molecular functions, leading to collaboration between CCN2 and FGFs during skeletal development.

DOI： 10.3390/ijms23158592

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Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described.

DOI： 10.3390/ijms23115887

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To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

DOI： 10.3390/microorganisms10051000

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Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

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OBJECTIVES: To clarify the role of cellular communication network factor 2/connective tissue growth factor (CCN2/CTGF) in periodontal tissue regeneration by investigating, the proliferative and tubulogenic responses of human endothelial cells obtained from the periodontal ligament to CCN2/CTGF. DESIGN: Endothelial cells were seeded on agar gel medium with or without 50 ng/mL recombinant CCN2/CTGF (rCCN2/CTGF) and cultured for 6 h. Cells were morphologically and phenotypically analyzed by immunofluorescent microscopy. A colorimetric assay was used to evaluate cell proliferation, and transmission electron microscopy (TEM) was used for ultrastructural analysis. RESULTS: The proliferation of endothelial cells was best promoted by rCCN2/CTGF at 50 ng/mL. In the control group, tube formation was not observed within 6 h. In contrast, endothelial cells seeded on the agar with 50 ng/mL rCCN2/CTGF clearly showed proliferation with network formation. Under a two-dimensional culture condition, a dense network of endothelial cells was not constructed on the plastic bottom. However, drastic morphological change was observed in the endothelial cells on the agar containing rCCN2/CTGF. The endothelial cells in the dense network were interconnected with each other and showed a tube-like structure. Tight junctions or adherens junctions were observed between the adjoining endothelial cells in the dense network. CONCLUSIONS: CCN2/CTGF was found to promote the proliferation and tubulogenesis of endothelial cells from the periodontal ligament. These results suggest that CCN2/CTGF may contribute to the regeneration of damaged periodontal tissue by activating the remaining endothelial cells.

DOI： 10.1016/j.archoralbio.2021.105279

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DOI： 10.1002/jcp.30348

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Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport protein 88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway.

DOI： 10.1016/j.bone.2021.116001

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The renin-angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT1R) and type 2 (AT2R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT1R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT1R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT1R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT1R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II-AT1R axis.

DOI： 10.3390/ijms22179204

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The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

DOI： 10.1016/j.bbrc.2021.06.013

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Cellular communication network factor (CCN) 3 is one of the classical members of the CCN family, which are characterized by common molecular structures and multiple functionalities. Although this protein was discovered as a gene product overexpressed in a truncated form in nephroblastoma, recent studies have revealed its physiological roles in the development and homeostasis of mammalian species, in addition to its pathological association with a number of diseases. Cartilage is a tissue that creates most of the bony parts and cartilaginous tissues that constitute the human skeleton, in which CCN3 is also differentially produced to exert its molecular missions therein. In this review article, after the summary of the molecular structure and function of CCN3, recent findings on the regulation of ccn3 expression and the roles of CCN3 in endochondral ossification, cartilage development, maintenance and disorders are introduced with an emphasis on the metabolic regulation and function of this matricellular multifunctional molecule.

DOI： 10.1007/s12079-021-00629-z

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Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

DOI： 10.1007/s12079-020-00600-4

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DOI： 10.1002/jcb.29680

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Cellular communication network factor 2 (CCN2) is a cysteine-rich secreted matricellular protein that regulates various cellular functions including cell differentiation. CCN2 is highly expressed under several types of mechanical stress, such as stretch, compression, and shear stress, in mesenchymal cells including chondrocytes, osteoblasts, and fibroblasts. In particular, CCN2 not only promotes cell proliferation and differentiation of various cells but also regulates the stability of mRNA of TRPV4, a mechanosensitive ion channel in chondrocytes. Of note, CCN2 behaves like a biomarker to sense suitable mechanical stress, because CCN2 expression is down-regulated when chondrocytes are subjected to excessive mechanical stress. These findings suggest that CCN2 is a mechano-sensing regulator. CCN2 expression is regulated by the activation of various mechano-sensing signaling pathways, e.g., mechanosensitive ion channels, integrin-focal adhesion-actin dynamics, Rho GTPase family members, Hippo-YAP signaling, and G protein-coupled receptors. This review summarizes the characterization of mechanoreceptors involved in CCN2 gene regulation and discusses the role of CCN2 as a mechano-sensing regulator of mesenchymal cell differentiation, with particular focus on chondrocytes.

DOI： 10.1016/j.jdsr.2020.07.001

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Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

DOI： 10.3390/ijms21207556

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DOI： 10.1016/j.job.2020.07.001

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At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

DOI： 10.15252/embr.201948462

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DOI： 10.1002/osi2.1076

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The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

DOI： 10.3390/cancers12051260

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To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

DOI： 10.3390/ijms21082769

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Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed.

DOI： 10.3390/ijms21051564

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In this study, we investigated the effect of CCN2 (cellular communication network factor 2), previously termed connective tissue growth factor, deposited in bone matrix on osteoclastogenesis and osteoblast differentiation. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-kappa B ligand (RANKL). NFATc1 and cathepsin K (CTSK) productions were more increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2 than the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. Interestingly, CTSK production was rather increased in KO osteocytes at 1 year after the injection. Finally, the combination of osteoblastic MC3T3-E1 and MLO-Y4 cells in rCCN2-containing bone matrix revealed the up-regulation of osteoblastic marker genes. These findings suggest that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation.

DOI： 10.1038/s41598-019-47285-3

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Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60 mW/cm2 intensity for 20 min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4 weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

DOI： 10.1007/s12079-018-0496-9

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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

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

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary BALB/c mouse chondrocytes caused enhanced cell growth and increased expression of <italic>Col2a1</italic>, <italic>Aggrecan</italic> and <italic>Sox9</italic>, but inhibited <italic>Col10a1</italic> expression. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of <italic>Aanat</italic>, <italic>Mt1</italic>, <italic>Mt2</italic> and <italic>Pthrp</italic> expression, followed by <italic>Sox9</italic> and <italic>Ihh</italic>. Furthermore, expression of the clock gene <italic>Bmal1</italic> was induced, while that of <italic>Per1</italic> was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of <italic>Aanat</italic> and modified the cyclic rhythm of <italic>Bmal1</italic>, <italic>Mt1</italic> and <italic>Mt2</italic>. In contrast, <italic>Mt1</italic> and <italic>Mt2</italic> showed different rhythms from <italic>Bmal1</italic> and <italic>Aanat</italic>, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

DOI： 10.1530/joe-19-0022

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DOI： 10.3390/medicines5040118

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A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.

DOI： 10.1002/jcp.26285

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Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

DOI： 10.3390/molecules23071517

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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

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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

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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

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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

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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

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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

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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

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DOI： 10.1007/978-1-4939-6430-7_6

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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. 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

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DOI： 10.1007/978-1-4939-6430-7_13

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DOI： 10.1007/978-1-4939-6430-7_14

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DOI： 10.1007/978-1-4939-6430-7_16

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DOI： 10.1007/978-1-4939-6430-7_4

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DOI： 10.1007/978-1-4939-6430-7_5

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CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927-937, 2016. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25377

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It is widely accepted that fibrosis is frequently observed in the gingiva of smokers. However, the mechanisms by which smoking results in pathological changes in periodontal tissue that lead to fibrosis are not entirely clear. Our former report showed that type I collagen synthesis was promoted by nicotine via CCN family protein 2 in human periodontal tissue cells. Here, we evaluated other aspects of nicotine function from a viewpoint of extracellular matrix (ECM) remodeling. Human gingival fibroblasts (n = 4) and periodontal ligament cells (n = 3) were isolated. The cells were treated with nicotine at a variety of concentrations for 12-48 h. Modulators of matrix remodeling were measured using enzyme-linked immunosorbent assays. Cell migration and morphology were also evaluated. As a result, following treatment with 1 mu g/ml nicotine, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta 1 production in both cell lysates and supernatants, and matrix metalloproteinases-1 production in cell lysates, were significantly increased (p &lt; 0.05). Compared to controls, cell migration was significantly inhibited (p &lt; 0.005) by nicotine in a time-dependent manner. Electron microscopic analysis revealed the presence of a number of vacuoles in nicotine-treated cells. These results indicate that nicotine not only impairs fibroblast motility, and induces cellular degenerative changes, but also alters ECM-remodeling systems of periodontal cells. Induction of matrix remodeling molecules, combined with type I collagen accumulation, may account for the molecular mechanism of nicotine-induced periodontal fibrosis.

DOI： 10.1007/s10266-014-0177-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOURNAL HARD TISSUE BIOLOGY  

Sorcin is a small calcium-binding protein that is widely expressed in many tissues. Sorcin regulates cardiac myocyte apoptosis by modulating mitochondrial Ca2+ cycling and regulates fibroblast cell cycling and apoptosis via Ca2+ signaling through the endoplasmic reticulum (ER). During endochondral ossification, some chondrocytes undergo apoptosis after their terminal differentiation; however, Sorcin's expression in these cells has not been characterized. Here we examined Sorcin's gene expression in the chondrocytes derived from mouse growth plate by reverse transcription PCR (RT-PCR), and its protein localization in the chondrocytes of femoral growth plate using immunohistochemistry. Sorcin protein was detected in the chondrocytes, and was particularly abundant in the cytoplasm and nuclei of proliferative zone chondrocytes and in the nuclei of hypertrophic chondrocytes. Apoptotic analysis of the growth plate revealed that many of the hypertrophic chondrocytes undergo the DNA fragmentation. We report for the first time the localization of Sorcin in the epiphyseal growth plate in which one of the apoptotic phenomenon was detected.

DOI： 10.2485/jhtb.25.57

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DOI： 10.1007/978-1-4939-6430-7_33

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Objectives: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.
Methods: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.
Results: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.
Conclusions: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

DOI： 10.3109/14397595.2016.1155255

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DOI： 10.1007/978-1-4939-6430-7_10

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DOI： 10.1007/978-1-4939-6430-7_8

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DOI： 10.1007/978-1-4939-6430-7_12

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© Springer Science+Business Media New York 2017. 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

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DOI： 10.1007/978-1-4939-6430-7_21

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DOI： 10.1007/978-1-4939-6430-7_25

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CCN family member 2 (CCN2) has been shown to promote the proliferation and differentiation of chondrocytes, osteoblasts, osteoclasts, and vascular endothelial cells. In addition, a number of growth factors and cytokines are known to work in harmony to promote the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification. Earlier we showed that CCN2 physically interacts with some of them, suggesting that multiple effects of CCN2 on various differentiation stages of chondrocytes may be attributed to its interaction with these growth factors and cytokines. However, little is known about the functional interaction occurring between CCN2 and other growth factors and cytokines in promoting chondrocyte proliferation and differentiation. In this study we sought to shed light on the binding affinities between CCN2 and other essential growth factors and cytokines known to be regulators of chondrocyte differentiation. Using the surface plasmon resonance assay, we analyzed the dissociation constant between CCN2 and each of the following: TGF-beta 1, TGF-beta 3, IGF-I, IGF-II, PDGF-BB, GDF5, PTHrP, and VEGF. We found a strong association between CCN2 and VEGF, as well as a relatively high association with TGF-beta 1, TGF-beta 3, PDGF-BB, and GDF-5. However, the sensorgrams obtained for possible interaction between CCN2 and IGF-I, IGF-II or PTHrP showed no response. This study underlines the correlation between CCN2 and certain other growth factors and cytokines and suggests the possible participation of such interaction in the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification.

DOI： 10.1007/s12079-015-0290-x

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CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a multi-potent factor for mesenchymal cells such as chondrocytes, osteoblasts, osteoclasts, and endothelial cells. CCN2 is also known as a modulator of other cytokines and receptors via direct molecular interactions with them. We screened additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as one of them. RANK is also known as TNF-related activation-induced cytokine (TRANCE) receptor, and its signaling plays a critical role in osteoclastogenesis. Notable affinity between CCN2 and RANK was confirmed by using surface plasmon resonance (SPR) analysis. In fact, CCN2 enhanced the RANK-mediated signaling, such as occurs in NF-kappa B, p38 and JNK pathways, in pre-osteoclastic RAW264.7 cells; whereas CCN2 had no influence on RANK RANK ligand (RANKL) binding. Moreover, CCN2 also significantly bound to osteoprotegerin (OPG), which is a decoy receptor of RANKL. Of note, OPG markedly inhibited the binding between CCN2 and RANK; and CCN2 canceled the inhibitory effect of OPG on osteoclast differentiation. These findings suggest CCN2 as a candidate of the fourth factor in the RANK/RANKL/OPG system for osteodastogenesis, which regulates OPG and RANK via direct interaction. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2014.12.058

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Many studies have reported that CCN family protein 2 (also known as connective tissue growth factor) induces fibrotic response in skeletal muscle, thus emphasizing the pathological role of CCN2 in muscle tissues. However, the physiological role of CCN2 in myogenesis is still unknown. This study clarified the CCN2 functions during myogenesis. Recombinant CCN2 (rCCN2) promoted proliferation and MyoD production in C2C12 cells and primary myoblasts, but inhibited myogenin production. In accordance with these findings, the gene expression levels of myosin heavy chain, which is a marker of terminally differentiated myoblasts and desmin, which is the main intermediate filament protein of muscle cells, were decreased by rCCN2 treatment. In vivo analyses with Ccn2-deficient skeletal muscle revealed decreased proliferating cell nuclear antigen (PCNA)/MyoD double positive cells and muscle hypoplasia. Consistent with this finding, myogenic marker genes and myotube formation were repressed in Ccn2-deficient myoblasts. The protein production of CCN2 was increased in C2C12 myoblasts treated with tumor necrosis factor-alpha, which is a pro-inflammatory cytokine, suggesting its role in muscle regeneration after inflammation. These findings indicate that CCN2 promotes proliferation and early differentiation but inhibits the terminal differentiation of myoblasts, thus suggesting that CCN2 plays a physiological role in myogenesis.

DOI： 10.1093/jb/mvu056

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Language：English   Publisher：PORTLAND PRESS LTD  

CCN family protein 2 (CCN2), also widely known as connective tissue growth factor (CTGF), is one of the founding members of the CCN family of matricellular proteins. Extensive investigation on CCN2 over decades has revealed the novel molecular action and functional properties of this unique signalling modulator. By its interaction with multiple molecular counterparts, CCN2 yields highly diverse and context-dependent biological outcomes in a variety of microenvironments. Nowadays, CCN2 is recognized to conduct the harmonized development of relevant tissues, such as cartilage and bone, in the skeletal system, by manipulating extracellular signalling molecules involved therein by acting as a hub through a web. However, on the other hand, CCN2 occasionally plays profound roles in major human biological disorders, including fibrosis and malignancies in major organs and tissues, by modulating the actions of key molecules involved in these clinical entities. In this review, the physiological and pathological roles of this unique protein are comprehensively summarized from a molecular network-based viewpoint of CCN2 functionalities.

DOI： 10.1042/CS20140264

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DOI： 10.1002/jbmr.2502

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In an attempt to find out a new molecular counterpart of CCN family protein 2 (CCN2), a matricellular protein with multiple functions, we performed an interactome analysis and found fibroblast growth factor (FGF) -1 as one of the candidates. Solid-phase binding assay indicated specific binding between CCN2 and FGF-1. This binding was also confirmed by surface plasmon resonance (SPR) analysis that revealed a dissociation constant (Kd) of 3.98 nM indicating strong molecular interaction between the two. RNA analysis suggested that both FGF-1 and CCN2 could be produced by chondrocytes and thus their interaction in the cartilage is possible. These findings for the first time indicate the direct interaction of CCN2 and FGF-1 and suggest the co-presence of these molecules in the cartilage microenvironment. CCN2 is a well-known promoter of cartilage development and regeneration, whereas the physiological and pathological role of FGF-1 in cartilage mostly remains unclear. Biological role of FGF-1 itself in cartilage is also suspected.

DOI： 10.1007/s12079-014-0232-z

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

CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a few genes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals. J. Cell. Biochem. 115: 854-865, 2014. (c) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.24728

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DOI： 10.1016/j.bone.2013.11.010

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The animal body is composed of a variety of cells and extracellular matrices that are organized and orchestrated in a harmonized manner to support life. Therefore, the critical importance of a comprehensive understanding of the molecular network surrounding and integrating the cells is now emphasized. The CCN family is a novel group of matricellular proteins that interact with and orchestrate a number of extracellular signaling and matrix molecules to construct and maintain living tissues. This family comprises six distinct members in mammals, which are characterized by a unique and conserved modular structure. These proteins are not targeted to limited and specific receptors to execute specific missions, but manipulate a vast number of biomolecules in the network by serving as a molecular hub at the center. The unified nomenclature, CCN, originates from a simple acronym of the three classical members, which helps us to avoid having any preconception about their pleiotropic and anonymous functional nature. In this review, after a brief summary of the general molecular concepts regarding the CCN family, new aspects of each member uncovered by recent research are introduced, which represent, nevertheless, only the tip of the iceberg of the profound functionality of these molecules. © 2013 Walter de Gruyter GmbH.

DOI： 10.1515/bmc-2013-0018

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Micro RNA (miRNA) is a small non-coding post-transcriptional RNA regulator that is involved in a variety of biological events. In order to specify the role of miRNAs in cartilage metabolism, we comparatively analyzed the expression profile of known miRNAs in chicken sternum chondrocytes representing early and late differentiation stages. Interestingly, none of the miRNAs displaying strong expression levels showed remarkable changes along with differentiation, suggesting their roles in maintaining the homeostasis rather than cytodifferentiation of chondrocytes. Among these miRNAs, miR-181a, which is known to play critical roles in a number of tissues, was selected and was further characterized. Human microarray analysis revealed remarkably stronger expression of miR-181a in human HCS-2/8 cells, which strongly maintained a chondrocytic phenotype, than in HeLa cells, indicating its significant role in chondrocytes. Indeed, subsequent investigation indicated that miR-181a repressed the expression of two genes involved in cartilage development. One was CCN family member 1 (CCN1), which promotes chondrogenesis; and the other, the gene encoding the core protein of aggrecan, a major cartilaginous proteoglycan, aggrecan. Based on these findings, negative feedback system via miR-181a to conserve the integrity of the cartilaginous phenotype may be proposed.

DOI： 10.1002/jcb.24556

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The 3'-untranslated region (UTR) is known to be a critical regulator of post-transcriptional events that determine the gene expression at the RNA level. The gene CCN1 is one of the classical members of the matricellular CCN family and is involved in a number of biological processes during mammalian development. In the present study, the 600-bp 3'-UTR of CCN1 was functionally characterized. Reporter gene analysis revealed that the entire 3'-UTR profoundly repressed gene expression in cis in different types of the cells, to which both the proximal and distal-halves of the 3'-UTR segments contributed almost equally. Deletion analysis of the 3'-UTR indicated a distinct functional element in the proximal half, whereas a putative target for microRNA-181s was predicted in silico in the distal half. Of note, the repressive RNA element in the proximal half was shown to be capable of forming a stable secondary structure. However, unexpectedly, a reporter construct with a tandem repeat of the predicted miR-181 targets failed to respond to miR-181a. In addition, the other major structured element predicted in the distal half was similarly characterized. To our surprise, the second element rather enhanced the reporter gene expression in cis. These results indicate the involvement of multiple regulatory elements in the CCN1 3'-UTR and suggest the complexity of the miRNA action as well as the 3'-UTR-mediated gene regulation.

DOI： 10.1007/s12079-013-0202-x

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Objective: Hypertrophy-like changes are often observed in chondrocytes during the development of osteoarthritis (OA). These changes play a crucial part in the OA-associated cartilage degradation and osteophyte formation. However, the pathogenesis leading to such changes is still unknown. In this study, we investigated the mechanism by which these hypertrophy-like changes are induced from the viewpoint of impaired glycolytic metabolism.
Methods: The effect of sodium fluoride (NaF) on glycolytic metabolism of cultured chondrocytes was confirmed by measurement of intracellular adenosine triphosphate (ATP) production. Translocation of phosphorylated Smad1/5/8 to the nucleus was evaluated by subcellular fractionation and Western blotting. Chondrocyte hypertrophy-like changes were investigated by real-time RT-PCR and Western blot analysis of differentiation markers.
Results: ATP production was dose-dependently decreased by NaF in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Interestingly, combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and matrix metalloproteinase13 (MMP13), which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions.
Conclusions: These findings suggest that decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate. Novel metabolic aspects of OA pathogenesis are indicated herein. (C) 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2013.01.013

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CCN family proteins 2 and 3 (CCN2 and CCN3) belong to the CCN family of proteins, all having a high level of structural similarity. It is widely known that CCN2 is a profibrotic molecule that mediates the development of fibrotic disorders in many different tissues and organs. In contrast, CCN3 has been recently suggested to act as an anti-fibrotic factor in several tissues. This CCN3 action was shown earlier to be exerted by the repression of the CCN2 gene expression in kidney tissue, whereas different findings were obtained for liver cells. Thus, the molecular action of CCN3 yielding its anti-fibrotic effect is still controversial. Here, using a general model of fibrosis, we evaluated the effect of CCN3 overexpression on the gene expression of all of the CCN family members, as well as on that of fibrotic marker genes. As a result, repression of CCN2 gene expression was modest, while type I collagen and α-smooth muscle actin gene expression was prominently repressed. Interestingly, not only CCN2, but also CCN4 gene expression showed a decrease upon CCN3 overexpression. These findings indicate that fibrotic gene induction is under the control of a complex molecular network conducted by CCN family members functioning together. © 2012 The International CCN Society.

DOI： 10.1007/s12079-012-0180-4

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DOI： 10.1371/journal.pone.0083545

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DOI： 10.1016/j.biochi.2012.10.016

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CCN2 plays a critical role in the development of mesenchymal tissues such as cartilage and bone, and the binding of CCN2 to various cytokines and receptors regulates their signaling. By screening a protein array, we found that CCN2 could bind to fibroblast growth factor receptors (FGFRs) 2 and 3, with a higher affinity toward FGFR2. We ascertained that FGFR2 bound to CCN2 and that the binding of FGFR2 to FGF2 and FGF4 was enhanced by CCN2. CCN2 and FGF2 had a collaborative effect on the phosphorylation of ERK and the differentiation of osteoblastic cells. The present results indicate the biological significance of the binding of CCN2 to FGFR2 in bone metabolism.
Structured summary of protein interactions:
FGFR2 binds to CCN2 by protein array (View interaction)
FGFR1OP binds to CCN2 by protein array (View interaction)
FGFR3 binds to CCN2 by protein array (View interaction) (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2012.10.038

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The intrinsic zone-specific properties of the menisci are determined by biomechanical environments. In this study, we examined mechanical stretch-dependent expression of multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, and investigated the role of CCN2 in meniscus cells. Uni-axial cyclic tensile strain (CTS) was applied using a STB-140 system. CTS-induced expression of CCN2 and a1(I) collagen (COL1A1) was assessed by quantitative real-time PCR analysis. The distribution of CCN2 and Smad2/3 in stretched cells was investigated by immunohistochemical analysis. Smad2/3-dependent CCN2 transactivation was measured by luciferase reporter assay. The relationship between Smad2/3 and CTS-induced CCN2 transcription was investigated by chromatin immunoprecipitation. CTS stimulated gene expression of CCN2 and COL1A1 in inner meniscus cells, but not in outer meniscus cells. Recombinant CCN2 increased COL1A1 expression only in inner meniscus cells. CCN2 synthesis and nuclear translocalization of phosphorylated Smad2/3 in inner meniscus cells were stimulated by CTS. The CCN2 promoter activity was synergistically enhanced by overexpressed Smad3 in stretched inner meniscus cells, but was not by Smad2. Chromatin immunoprecipitation revealed that CTS increased the association between Smad3 and the Smad-binding element on the CCN2 proximal promoter in inner meniscus cells. Our results suggest that stretch-induced CCN2 may have a crucial role in regulating COL1A1 expression in the inner meniscus. (c) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:17381745, 2012

DOI： 10.1002/jor.22142

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CCN2 is one of the representative members of the CCN family, a group of proteins that orchestrate the extracellular signaling network. As anticipated by the original name, connective tissue growth factor, this molecule promotes the growth and development of mesenchymal tissues, including bone and cartilage. Indeed, CCN2 is required for the proper development of the orofacial region, which requirement is typically suggested by the frequent emergence of cleft palate in CCN2-null mice. The significant contribution of CCN2 to mandibular morphogenesis and tooth germ development has also been indicated. Of note, CCN2 functions not only during development, but also later in life, as it is a critical promoter of physiological and pathological tissue remodeling, the latter of which denotes fibrotic reconstruction of tissue. In addition to its involvement in fibrotic disorders in a variety of organs, CCN2 has been also reported to be a mediator of periodontal fibrosis caused by several factors including smoking. Based on these cumulative findings, the utility of CCN2 to accelerate oral tissue regeneration by a harmonized remodeling process is discussed herein, together with regulation of the gene expression and molecular function of CCN2 as a therapeutic strategy against periodontal fibrosis. © 2012 Japanese Association for Dental Science.

DOI： 10.1016/j.jdsr.2012.02.002

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Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

DOI： 10.1242/jcs.101956

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DOI： 10.1242/jcs.101956.

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The CCN family of proteins consists of six members with conserved structural features. These proteins play several roles in the physiology and pathology of cells. Among the pathological roles of the CCN family, one of the most important and controversial ones is their role in the expansion and metastasis of cancer. Up to now a number of reports have described the possible role of each CCN family member independently. In this study, we comprehensively analyzed the roles of all six CCN family members in cell growth, migration and invasion of breast cancer cells in vitro and in vivo. As a result, we found the CCN2/CCN3 ratio to be a parameter that is associated with the metastatic phenotype of breast cancer cells that are highly metastatic to the bone. The same analysis with cell lines from oral squamous carcinomas that are not metastatic to the bone further supported our notion. These results suggest the functional significance of the interplay between CCN family members in regulating the phenotype of cancer cells.

DOI： 10.1007/s12079-011-0133-3

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

CCN2 (also known as connective tissue growth factor) interacts with several growth factors involved in endochondral ossification via its characteristic four modules and modifies the effect of such growth factors. Presently we investigated whether CCN2 interacts with fibroblast growth factor 2 (FGF2). Solid-phase binding assay, immunoprecipitation-Western blot analysis, and surface plasmon resonance (SPR) spectroscopy revealed that the C-terminal module of CCN2 (CT) directly bound to FGF2 with a dissociation constant of 5.5 nM. Next, we examined the combinational effects of CCN2 and FGF2 on the proliferation of and matrix metalloproteinase (MMP)-9 and -13 productions by cultured chondrocytes. FGF2 promoted not only the proliferation but also the production of MMP9 and -13, however, combined of FGF2 with CT module nullified the enhancement of both MMP productions and proliferation. To clarify the mechanism, we investigated the binding of CCN2 or its CT module to FGF receptor 1. As a result, we found that CCN2 bound to FGF receptor 1 with a dissociation constant of 362 nM, whereas the CT module did not. In addition, when we tested FGF signaling in chondrocytic HCS-2/8 cells stimulated by the combination of FGF2 with CT module, the level of ERK1/2, p38 MAPK, and c-Jun N-terminal kinase phosphorylation was decreased compared with that found with FGF2 alone. These findings suggest that CCN2 may regulate the proliferation and matrix degradation of chondrocytes by forming a complex with FGF2 as a novel modulator of FGF2 functions. (Endocrinology 152: 4232-4241, 2011)

DOI： 10.1210/en.2011-0234

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Purpose of the research: Ligneous gingivitis is a rare disease characterized by nodular gingival enlargement secondary to fibrin deposits induced by micro-injury in the gingiva, which disorder results from plasminogen (PLG) deficiency. Although none have investigated the association of wound healing factors with ligneous gingivitis. In this study, in addition to a histopathologic examination of ligneous gingivitis in a case of type I PLG deficiency, we further present data showing the effect of wound healing factors in association with fibrin in vitro to clarify the pathobiology of ligneous gingivitis in PLG-deficient patients. Principle results: Immunohistochemical analysis revealed that transforming growth factor (TGF)-β1, connective tissue growth factor/CCN2 (CCN2), and endothelin-1 (ET-1) had accumulated in the extracellular matrix around the epithelial and fibroblastic cells near the fibrin deposition. Consistent with these results, fibrin and TGF-β1 synergistically up-regulated CCN2 and ET-1 gene expression in human dermal fibroblasts. Major conclusions: Fibrin plays a vicious role in ligneous gingivitis pathobiology by up-regulating CCN2 and ET-1 expression through the TGF-β signaling pathway. © 2011 Japanese Stomatological Society.

DOI： 10.1016/S1348-8643(11)00025-5

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CCN family proteins play diverse roles in many aspects of cellular processes such as proliferation, differentiation, adhesion, migration, angiogenesis and survival. In the bone tissue of vertebrate species, the expression of most CCN family members has been observed in osteoblasts. However, their spatial and temporal distributions, as well as their functions, are still only partially understood. In this study, we evaluated the localization of CCN family members in skeletal tissue in vivo and comparatively analyzed the gene expression patterns and functions of the members in murine osteoblasts in primary culture. Immunofluorescent analyses revealed that the CCN family members were differentially produced in osteoblasts and osteocytes. The presence of all Ccn transcripts was confirmed in those osteoblasts. Among the members, CCN1, CCN2, CCN4 and CCN5 were found in osteocytes. CCN4 and CCN5 were distributed in osteocytes located inside of bone matrix as well. Next, we investigated the expression pattern of Ccn family members during osteoblast differentiation. Along with differentiation, most of the members followed proper gene expression patterns: whereas, Ccn4 and Ccn5 showed quite similar patterns. Furthermore, we evaluated the effects of CCN family members on the osteoblastic activities by using recombinant CCN proteins and RNA interference method. Five members of this family displayed positive effects on osteoblast proliferation or differentiation. Of note, CCN3 drastically inhibited the osteoblast activities. Each Ccn specific siRNA could modulate osteoblast activities in a manner expected by the observed effect of respective recombinant CCN protein. In addition, we found that extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase pathways were critically involved in the CCN family member-mediated modification of osteoblast activities.
Collectively, all Ccn family members were found to be differentially expressed along with differentiation and therefore could participate in progression of the osteoblast lineage. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.06.033

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Identification and characterization of local molecules directing the differentiation of chondrocytes to either transient or permanent cartilage are major issues in cartilage biology. Here, we found CCN family protein 3 (CCN3) was abundantly produced in rat developing epiphyseal cartilage. Evaluations in vitro showed that CCN3 repressed epiphyseal chondrocyte proliferation, while promoting matrix production in multiple assays performed. Furthermore, CCN3 enhanced the articular chondrocytic phenotype; whereas it repressed the one representing endochondral ossification. Additionally, the phenotype of growth plate chondrocytes and chondrogenic progenitors also appeared to be affected by CCN3 in a similar manner. These findings suggest a significant role of CCN3 in inducing chondrocytes to articular ones during joint formation. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2011.08.024

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Chondrocytes forming articular cartilage are embedded in a vast amount of extracellular matrix having physical stiffness and elasticity, properties that support the mechanical load from bones and enable the flexible movement of synovial joints. Unlike chondrocytes that conduct the growth of long bones by forming the growth plate, articular chondrocytes show suppressed cell proliferation, unless these cells are exposed to pathological conditions such as mechanical overload. In the present study, we found that one of the members of the CCN family, CCN3, was significantly expressed in chondrocytes isolated from the epiphyseal head in developing rat synovial joints. Evaluation of the effect of recombinant CCN3 on those chondrocytes revealed that CCN3 promoted proteoglycan synthesis, whereas this factor repressed the proliferation of the same cells. These results suggest a critical role for CCN3 in the regulation of the biological properties of articular chondrocytes.

DOI： 10.1007/s12079-011-0135-1

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CCN2, a classical member of the CCN family of matricellular proteins, is a key molecule that conducts cartilage development in a harmonized manner through novel molecular actions. During vertebrate development, all cartilage is primarily formed by a process of mesenchymal condensation, while CCN2 is induced to promote this process. Afterwards, cartilage develops into several sub-types with different fates and missions, in which CCN2 plays its proper roles according to the corresponding microenvironments. The history of CCN2 in cartilage and bone began with its re-discovery in the growth cartilage in long bones, which determines the skeletal size through the process of endochondral ossification. CCN2 promotes physiological developmental processes not only in the growth cartilage but also in the other types of cartilages, i.e., Meckel's cartilage representing temporary cartilage without autocalcification, articular cartilage representing hyaline cartilage with physical stiffness, and auricular cartilage representing elastic cartilage. Together with its significant role in intramembranous ossification, CCN2 is regarded as a conductor of skeletogenesis. During cartilage development, the CCN2 gene is dynamically regulated to yield stage-specific production of CCN2 proteins at both transcriptional and post-transcriptional levels. New functional aspects of known biomolecules have been uncovered during the course of investigating these regulatory systems in chondrocytes. Since CCN2 promotes integrated regeneration as well as generation (=development) of these tissues, its utility in regenerative therapy targeting chondrocytes and osteoblasts is indicated, as has already been supported by experimental evidence obtained in vivo.

DOI： 10.1007/s12079-011-0123-5

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The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mchm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

DOI： 10.1172/JCI44957

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

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates alpha 1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.04.138

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

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF-kappa B ligand (GST-RANKL), and a combination of recombinant CCN2 (rCCN2) and GST-RANKL significantly enhanced tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cell formation compared with GST-RANKL alone. Therefore, we suspected the involvement of CCN2 in cell-cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real-time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid-phase binding assay of CCN2 and dendritic cell-specific transmembrane protein (DC-STAMP), which is involved in cell-cell fusion. The results showed that CCN2 induced and interacted with DC-STAMP. Furthermore, GST-RANKL-induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC-STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC-STAMP. (C) 2011 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.222

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

CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3&apos;-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.01.034

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

CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine-cytosine (GC)-rich region of approximately 200 bp near the 5'-end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility-shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5'-end, but did not do so at the 3'-end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC-rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual-functionality of the mRNA segment in the eat ! open reading frame, which segment acts as a cis-element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein. J. Cell. Biochem. 111: 1607-1618, 2010. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jcb.22894

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

The process of endochondral ossification is strictly regulated by a variety of extracellular and intracellular factors. Recently, it has become recognized that specific miRNAs are involved in this process by regulating the expression of the relevant genes at the post-transcriptional level. In this present study we obtained the first evidence of the involvement of a specific micro RNA (miRNA) in the regulation of the chondrocyte phenotype during late stages of differentiation. By use of the microarray technique, miR-1 was identified as this miRNA, the expression of which was most repressed upon hypertrophic differentiation. Transfection of human chondrocytic HCS-2/8 cells and chicken normal chondrocytes with miR-1 led to repressed expression of aggrecan, the major cartilaginous proteoglycan gene. Therefore, miR-1 was found to be involved in the regulation of the chondrocytic phenotype and thus to play an important role in chondrocytes during the late stage of the differentiation process, maintaining the integrity of the cartilage tissue. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.10.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) consists of 4 conserved modules that are highly interactive with a number of biomolecules. With such interaction, CCN2 exerts multiple functions by forming an extracellular information network. In the present study, we screened for dodecapeptide sequences that bound to each module of human CCN2 by using a bacteriophage display library. Thereafter, consensus amino acid sequences for the binding to individual modules were extracted in silico and utilized to design anchor peptide aptamers that would facilitate the interaction between CCN2 and other molecules. Direct binding of a few peptides to CCN2 was confirmed by surface plasmon resonance analysis. Subsequent biological assay indicated that one such peptide was capable of promoting the proliferation of CCN2-producing chondrocytic cells. This cell biological activity was found to be sequence specific and CCN2 dependent. Since CCN2/CTGF was shown to be effective in articular cartilage/bone regeneration in vivo, utility of such peptide aptamers in CCN2-associated regenerative therapeutics is suggested herein. (C) 2010 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2010.04.021

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

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

DOI： 10.1007/s12079-009-0067-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

Since fibrosis is observed in smokers&apos; gingiva, it was hypothesized that fibrosis was caused by nicotine in the periodontium. Therefore, in this study, we investigated the effects of nicotine on the induction of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF), in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells. With 1 mu g/mL nicotine, vacuolization and attenuated proliferation were observed. Interestingly, 1 mu g/mL nicotine increased the production of CCN2/CTGF protein in both cells without increasing mRNA expression. Furthermore, type I collagen mRNA and protein were also increased and were significantly blocked by a CCN2/CTGF neutralizing antibody. This is the first report to describe a relationship between nicotine and CCN2/CTGF in periodontal tissue cells. Analysis of our data also indicated that nicotine was cytotoxic, while it increased CCN2/CTGF and, eventually, type I collagen production. These findings suggest that periodontal fibrosis can be promoted by nicotine from smoking via effects on CCN2/CTGF.

DOI： 10.1177/0022034509353403

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

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown Irpl in chondrocytic cells and obtained findings indicating a critical role therein. As a result of IrpI knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axing, which is known to be induced by activation of the WNT/beta-catenin (beta cat) signaling pathway. Thereby, we found that Axing promoter activity was enhanced in the Irpl knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3 beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the Irp1 knockdown cells. When the phosphorylation of PKC zeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1. J. Cell. Physiol. 222: 138-148, 2010. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcp.21930

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG BERLIN  

In this chapter, we introduce a new trend in the field of CCN proteins research, that is, the yin/yang effects of CCN2 and CCN3 and the mutual regulation of ccn2 and ccn3 gene expression by these two proteins. These findings point out the need for a more thorough investigation of functional interactions between CCN proteins in normal and pathological conditions

DOI： 10.1007/978-90-481-3779-4_8

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG BERLIN  

CCN2/CTGF, previously known as Connective Tissue Growth Factor, is a crucial regulator of extra-cellular matrix (ECM), which promotes ECM synthesis and stabilization. As their family name clearly implies, matrix metalloproteases (MMPs) are also localized in the ECM, where they function as proteases, modulating cell signaling by cleaving proteins such as matrix proteins, growth factors and growth factor receptors. Strong expression of CCN2/CTGF in chondrocytic cells occurs through transcription enhancer dominant in chondrocytes (TRENDIC). Matrix metalloprotease-3 (MMP3) is a novel TRENDIC-binding transcription factor for CCN2/CTGF expression. First, MMP3 cDNA was cloned as a TRENDIC-binding factor by Southwestern screening. The interaction between MMP3 and TRENDIC was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by transfected MMP3, whereas a TRENDIC mutant for the promoter lost the response. In addition, the knockdown of MMP3 suppressed CCN2/CTGF expression. Cytochemical and histochemical analyses demonstrated that MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 was observed in 30 min after the addition, and six putative nuclear localization signals were found in MMP3. These results indicated a novel trans-activation mechanism of CCN2/CTGF by the nuclear translocation of MMP3 through binding with TRENDIC in chondrocytes. Although MMPs historically had been recognized as a protease for extra-cellular proteins, this study indicated that it also stimulates ECM synthesis through CCN2/CTGF trans-activation. This novel regulatory role of the ECM may contribute to understanding the mechanism of not only the development, but also the pathogenesis of arthritis fibrosis and periodontitis.

DOI： 10.1007/978-90-481-3779-4_19

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Other Link： http://orcid.org/0000-0002-9600-4430

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG BERLIN  

CCN2/CTGF is a multifunctional molecule that has been shown to play a central role in chondrocyte differentiation. During this process, the expression of ccn2 is tightly regulated to confer a maximal level at prehypertrophic - hypertrophic stages, in which the 3'-untranslated region (UTR) of the mRNA is critically involved in mediating its post-transcriptional regulation. In our previous studies, we found that a 40-kDa protein binding specifically to an RNA cis-element, 3'-100/50, in the 3'-UTR of the chicken ccn2 mRNA regulated the intracellular stability of the mRNA. The interaction of this 40-kDa protein with 3'-100/50 was enhanced in proliferating chondrocytes, in which ccn2 mRNA is rapidly degraded; whereas a prolonged half life of ccn2 mRNA is observed in hypertrophic chondrocytes, where the interaction of the 40 kDa-protein and 3'-100/50 is diminished. Collectively, the data suggested that this 40-kDa protein acts as a ccn2-specific mRNA destabilizer during chondrocyte differentiation.
In this present study we finally identified this 40-kDa protein as nucleophosmin (NPM)/B23. NPM is a nuclear-cytoplasmic shuttling protein that is characterized by its multiple functionality. This protein is known to be a histone chaperone, a regulator of ribosomal RNA transcription, as well as an RNA-binding post-transcriptional regulator of gene expression. In our hands, direct binding of NPM to 3'-100/50 was confirmed not only by RNA EMSA and UV crosslinking assays, but also by RNA immunoprecipitation analysis. By using recombinant chicken NPM, we could successfully reconstitute the post-transcriptional regulation of ccn2 by NPM in vitro and found that this regulation was more robust in chondrocytes than in fibroblasts. Furthermore, siRNA-mediated gene silencing of NPM in vivo clearly showed enhanced ccn2 gene expression and a prolonged half life of the ccn2 mRNA, confirming the functional property of NPM as a specific destabilizer of the ccn2 mRNA in living cells.
The 5'-100/50 element, a target of NPM, is evolutionally conserved among vertebrate species. Therefore, we consider NPM to be a critical post-transcriptional regulator of ccn2 acting via 3'-UTR during endochondral ossification and possibly, in other physiological and pathological states as well.

DOI： 10.1007/978-90-481-3779-4_4

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Other Link： http://orcid.org/0000-0002-9600-4430

Language：Japanese   Publisher：先端医学社  

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Other Link： http://search.jamas.or.jp/link/ui/2011089282

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried Out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, its a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type I repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan. and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding in vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP-rVWC peptide effectively enhanced tire production and release of aggrecan compared with the rTSP-rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC Modules. and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

DOI： 10.1042/BJ20081991

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

Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro.
Cells were isolated from normal periodontal tissues and cultured in Dulbecco&apos;s modified Eagle&apos;s minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco&apos;s modified Eagle&apos;s minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy.
In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization.
The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

DOI： 10.1111/j.1600-0765.2008.01093.x

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

We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3'-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2009.02.025

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

Both CCN family 2/connective tissue growth factor (CCN2/CTGF) and bone morphogenetic protein (BMP)-2 play an important role in cartilage metabolism. We evaluated whether or not CCN2 would interact with BMP-2, and examined the combination effect of CCN2 with BMP-2 (CCN2-BMP-2) on the proliferation and differentiation of chondrocytes. Immunoprecipitation-western blotting analysis, solid-phase binding assay and surface plasmon resonance (SPR) spectroscopy showed that CCN2 directly interacted with BMP-2 with a dissociation constant of 0.77 nM as evaluated by SPR. An in vivo study revealed that CCN2 was co-localized with BMP-2 at the pre-hypertrophic region in the E18.5 mouse growth plate. Interestingly, CCN2-BMP-2 did not affect the BMP-2/CCN2-induced phosphorylation of p38 MAPK but caused less phosphorylation of ERK1/2 in cultured chondrocytes. Consistent with these results, cell proliferation assay showed that CCN2-BMP-2 stimulated cell growth to a lesser degree than by either CCN2 or BMP-2 alone, whereas the expression of chondrocyte marker genes and proteoglycan synthesis, representing the mature chondrocytic phenotype, was increased collaboratively by CCN2-BMP-2 treatment in cultured chondrocytes. These findings suggest that CCN2 may regulate the proliferating and differentiation of chondrocytes by forming a complex with BMP-2 as a novel modulator of BMP signalling.

DOI： 10.1093/jb/mvn159

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

Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O(2)) of HCS-2/8 cells increased VEGF mRNA levels by similar to 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1 alpha mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1 alpha activity. In fact, HIF-1 alpha was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 Mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2008.08.125

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

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members. and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, We conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins oil chondrocyte proliferation, differentiation, and the expression of chondrocyte-associated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes ill vitro. whereas CcO expression drastically increased. In contrast, the addition Of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes. whereas decreasing, the CcO expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, Such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

DOI： 10.1359/JBMR.080615

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3' untranslated region (3'-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

DOI： 10.1128/MCB.00495-08

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

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated Upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning Microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.06.089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO LTD  

Objectives: CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo.
Methods: Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [3 H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis.
Results: CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type 11 and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF.
Conclusions: These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction Of Elastic cartilage. (C) 2007 Ostecarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2007.11.001

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

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However,there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP6001 25 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximicle completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by I 2-0-tetradecanoylphorbol I 3-acetate (TPA). These resu Its suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and thatcle novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.

DOI： 10.1002/jeb.21724

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

Background: Mandibular bone destruction is a frequent occurrence in oral squamous cell carcinoma. However, the relationship between the bone destruction and associated factors is unclear. Here, the role and diagnostic utility of connective tissue growth factor (CCN2) in bone destruction of the mandible was investigated. Patients and Methods: The production of CCN2 was explored by using immunohistochemistry on paraffin-embedded tissues from 20 cases of mandibular squamous cell carcinoma. The effect of CCN2 on osteoclastogenesis was examined in vitro by using total bone marrow cell populations from male mice. Results: Immunohistochemical analysis showed that CCN2-positive signals were closely associated with destructive invasion of the mandible by oral squamous cell carcinomas. Consistent with these results, recombinant human CCN2 (rCCN2) stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cell formation in vitro. Conclusion: CCN2 can be considered a diagnostic marker and target for treatment in oral osteolytic mandibular squamous cell carcinoma.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique molecule that promotes the entire endochondral ossification process and regeneration of damaged articular cartilage. Also, CCN2 has been shown to enhance the adhesion and migration of bone marrow stromal cells as well as the growth and differentiation of osteoblasts; hence, its utility in bone regeneration has been suggested. Here, we evaluated the effect of CCN2 on the regeneration of an intractable bone defect in a rat model. First, we prepared two recombinant CCN2s of different origins, and the one showing the stronger effect on osteoblasts in vitro was selected for further evaluation, based on the result of an in vitro bioassay. Next, to obtain a sustained effect, the recombinant CCN2 was incorporated into gelatin hydrogel that enabled the gradual release of the factor. Evaluation in vivo indicated that CCN2 continued to be released at least for up to 14 days after its incorporation. Application of the gelatin hydrogel-CCN2 complex, together with a collagen scaffold to the bone defect prepared in a rat femur resulted in remarkable induction of osteoblastic mineralization markers within 2 weeks. Finally, distinct enhancement of bone regeneration was observed 3 weeks after the application of the complex. These results confirm the utility of CCN2 in the regeneration of intractable bone defects in vivo when the factor is incorporated into gelatin hydrogel.

DOI： 10.1089/ten.tea.2007.0167

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested.

DOI： 10.1128/MCB.01288-07

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

Background: Connective tissue growth factor (CTGF) has been recently reported as a mediator of myocardial fibrosis; however, the significance of plasma CTGF concentration has not been evaluated in patients with heart failure. The aim of this study was to investigate the clinical utility of plasma CTGF concentration for the diagnosis of heart failure.
Methods and results: We evaluated fifty-two patients with chronic heart failure. The plasma concentration of CTGF and other markers of fibrosis were assessed and compared with clinical and echocardiographic data. Plasma CTGF was significantly elevated in symptomatic patients in proportion to their NYHA classes and was significantly correlated with plasma brain natriuretic peptide (BNP) concentration (r=0.395, P&lt;0.01). Plasma CTGF was also correlated with plasma transforming growth factor beta (TGF-beta) (r=0.512, P&lt;0.01), matrix metalloproteinase (MMP)-2 (r=0.391, P&lt;0.05) and tissue inhibitor of MMP (TIMP)-2 (r=0.354, P&lt;0.05) concentrations. Interestingly, plasma CTGF was correlated with E/E' value evaluated by tissue Doppler echocardiography (r=0.593, P=0.012), but not with systolic function and left ventricular mass.
Conclusion: Our study suggests that plasma CTGF concentration is a novel diagnostic marker for cardiac dysfunction and may provide additional specific information about myocardial fibrosis in chronic heart failure patients. (C) 2008 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejheart.2008.02.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

Mechanical stress plays a key role in the pathogenesis of cartilage destruction seen in osteoarthritis (OA). We investigated the effect of cyclic tensile stress (CTS) on the anabolic and catabolic gene expression of rat cultured normal chondrocytes using the Flexercell strain unit. The effects of interleukin (IL)-4, a chondroprotective cytokine, on the changes in gene expression induced by CTS were also investigated. CTS (7% elongation at 0.5 Hz) for 24 h did not affect the expression of aggrecan and type 11 collagen, whereas CTS significantly upregulated matrix metalloproteinase (MMP)-13 and cathepsin B mRNA expression by chondrocytes. IL-1 beta expression was also significantly upregulated by CTS up to 12 h. The upregulation of MMP-13 was observed at 3 h, which was earlier than that of IL-1 beta. Furthermore, pre-treatment with IL-4 (10 ng/ml) suppressed both MMP-13 and cathepsin B induction by mechanical stress, as well as CTS-induced IL-1 beta expression. Our results suggest that IL-4 might have a therapeutic value in the treatment of OA by downregulation of mechanical stress-induced MMP-13 and cathepsin B expression by chondrocytes.

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

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E-2 without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.11.089

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

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.11.155

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COGNIZANT COMMUNICATION CORP  

Multiple roles have been already recognized for CCN2 in cartilage development and regeneration. However, the effects of CCN2 on bone regeneration remain to be elucidated. In this study, the utility of CCN2 on bone regeneration was examined in vitro and in vivo in combination with hydroxyapatite (HAp) as a scaffold. Human bone marrow stromal cells (hBMSCs) were isolated from human iliac bone marrow aspirates of healthy donors and expanded, and the effects of CCN2 on their proliferation and migration were examined in vitro. The proliferation of hBMSCs on a plastic or HAp plate was significantly enhanced by CCN2. Moreover, the migration of hBMSCs also dramatically increased by CCN2. Interestingly, a C-terminal signal modular fragment of CCN2 (CT-module) also enhanced the cell proliferation and migration as efficiently as the full-length CCN2. Next, in order to estimate the effect of CCN2 on the migration and survival of hBMSCs and bone formation inside the HAp scaffold in vivo, two experiments were performed. First, the porous HAp carrier was cultured with hBMSCs for a week, and the cell-scaffold hybrid was transplanted with or without CCN2 subcutaneously into immunocompromised mice. CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HAp within 4 weeks after transplantation. Second, the porous HAp carrier with or without CCN2 was directly implanted into bone defects within a rabbit mandible, and bone regeneration inside was evaluated. As a result, CCN2 efficiently induced the cell invasion and bone formation inside the porous HAp scaffold. These findings suggest that CCN2 and its CT-module fragment could be useful for regeneration and reconstruction of large-scale bone defects.

DOI： 10.3727/000000008783907143

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

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.

DOI： 10.3233/BIR-2008-0478

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HISTOCHEMICAL SOC INC  

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metal loproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

DOI： 10.1369/jhc.7A7263.2007

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

Cell attachment is a crucial step in tissue regeneration. In this study, human bone marrow stromal cells (hBMSCs) were isolated, and the effects of CCN2 on their attachment were examined. CCN2 significantly enhanced the hBMSC attachment, and this enhanced cell attachment was mainly regulated by the C-terminal module of CCN2. This enhancement was negated by the anti-integrin alpha(v)beta(3) antibody and p38 MAPK inhibitor, and phosphorylation of p38 MAPK was detected upon the enhanced cell attachment mediated by CCN2. We thus conclude that CCN2 enhances hBMSC attachment via integrin-p38 MAPK signal pathway. Enhanced hBMSC attachment on hydroxyapatite plates by CCN2 further indicated the utility of CCN2 in bone regeneration. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.03.052

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein - coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

DOI： 10.1161/HYPERTENSIONAHA.106.077537

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

CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.

DOI： 10.1111/j.1742-4658.2007.05709.x

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Language：English  

The CCN family is a novel class of extracellular signal modulators that has been recently established. Typical members are composed of four conserved modules connected tandem, each of which is rich in cysteines and highly interactive with other molecules. The mammalian CCN family consists of six members, most of which have been described as multifunctional factors for the developmental process of mesenchymal tissue including blood vessel formation/induction. Particularly, the angiogenic properties of the three classical members, CCN1, 2 and 3 have so far been characterized, and their physiological and pathological significance has thus been indicated. Recent research has uncovered a unique mechanism regarding these proteins in promoting and/or modulating developmental, physiological and pathological angiogenic events. Namely, CCN proteins exert their ability to drive angiogenesis, not by stimulating a particular behavior of a particular type of cells, but by manipulating the cell communication networks that integrate most of the associated molecules/cells toward angiogenesis. In this article, the role of the CCN proteins in a variety of angiogenic events as an organizer of microenvironmental cell society is comprehensively described, together with a brief summary of the recent findings on each CCN family member relevant to angiogenesis including cardiovascular development and diseases. © 2006 Springer Science + Business Media B.V.

DOI： 10.1007/s10456-006-9058-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Connective tissue growth factor (CTGF/CCN2) plays a critical role in endochondral bone formation; however, CCN2 also promotes angiogenesis and bone metastasis in breast cancer. Chondrocytic HCS-2/8 cells and breast cancer MDA231 cells produce over 6 times more CCN2 than any other cell type. In this study, we demonstrate that these cell lines employ different transcriptional strategies for ccn2 gene induction. Four tandem copies of the dominant transcriptional enhancer in chondrocytes (4 x TRENDIC) were chimerically connected to an SV40 pro-moter-luciferase construct and subsequently analyzed. The enhancement of the promoter activity by 4 x TRENDIC was greater in the HCS-2/8 cells (7-fold) than in the other 4 cell lines (3-4 fold). The TRENDIC-binding protein complex was detected at a higher signal in the HCS-2/8 cells than in the other cell lines. In addition, the HCS-2/8 nuclear factors strongly targeted not only TRENDIC, but also the previously reported basal control element and a novel enhancer element in the ccn2 promoter. In contrast, high-level ccn2 gene induction in MDA231 cells was largely dependent on Smad signaling through the Smad-binding element in the ccn2 promoter. Based on these results, we propose a model of differential transcription of the ccn2 gene between the chondrocytic cell line and the breast cancer cell line, and therefore imply that these cells utilize distinct transcriptional strategies to obtain the enhanced CCN2 production that is not observed in other types of cells. (c) 2007 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2006.12.006

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Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

Our bones mostly develop through a process called endochondral ossification. This process is initiated in the cartilage prototype of each bone and continues through embryonic and postnatal development until the end of skeletal growth. Therefore, the central regulator of endochondral ossification is the director of body construction, which is, in other words, the determinant of skeletal size and shape. We suggest that CCN2/CTGF/Hcs24 (CCN2) is a molecule that conducts all of the procedures of endochondral ossification. CCN2, a member of the CCN family of novel modulator proteins, displays multiple functions by manipulating the local information network, using its conserved modules as an interface with a variety of other biomolecules. Under a precisely designed four-dimensional genetic program, 002 is produced from a limited population of chondrocytes and acts on all of the mesenchymal cells inside the bone callus to promote the integrated growth of the bone. Furthermore, the utility of CCN2 as regenerative therapeutics against connective tissue disorders, such as bone and cartilage defects and osteoarthritis, has been suggested. Over the years, the pathological action of CCN2 has been suggested. Nevertheless, it can also be regarded as another aspect of the physiological and regenerative function of CCN2, which is discussed as well.

DOI： 10.1016/S0074-7696(07)57001-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2. (c) 2006 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2006.07.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BONE & MINERAL RES  

Introduction: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related.
Materials and Methods: A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated.
Results: Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA), a stimulator of PKC, upregulated it.
Conclusions: CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

DOI： 10.1359/JBMR.060416

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

CCN2/connective tissue growth factor (CCN2/CTGF) is known to promote both the proliferation and differentiation of chondrocytes, which actions are mediated by ERK and p38 MAPK, respectively. In this study, we first re-evaluated the involvement of multiple MAPKs therein and found that JNK also mediated such CCN2 signals. Thereafter, we further analyzed the roles of upstream kinases. The involvement of PKC, PI3K and PKA in the CCN2 signaling to promote the maturation, proliferation and terminal differentiation of a human chondrocytic cell line, HCS-2/8 and rabbit primary growth cartilage cells was investigated. As a result, the PKC inhibitor calphostin C repressed all of the effects of CCN2, which were represented by increased synthesis of DNA and proteoglycans and the display of alkaline phosphatase activity. In addition, evaluation of the effect of the PI3K inhibitor wortmannin disclosed the contribution of PI3K in transducing CCN2 signals to promote chondrocyte hypertrophy. This signal was known to be mediated by PKB, which was translocated into the nucleus upon CCN2 stimulation. Of note, calphostin C showed inhibitory effects on the activation of p38 MAPK, ERK and also PKB, whereas it exerted no effect on JNK activation. These results suggest that PKC is a driver of multiple signal transducing kinases that promote the proliferation and differentiation of chondrocytes. The requirement of PI3K in transmitting the signal for terminal differentiation and PKC-independent signaling pathways for the promotion of chondrocytic growth and differentiation, which was mediated by JNK, were also uncovered. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2005.11.016

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

Low density lipoprotein receptor (LDLR)-related protein 1 (LRP1/CD91) is one of the receptors of CCN2 that conducts endochondral ossification and cartilage repair. LRP1 is a well-known endocytic receptor, but its distribution among chondrocytes remains to be elucidated. We herein demonstrate for the first time that the distribution of LRP1 in chondrocytes except for hypertrophic chondrocytes in vivo and in vitro. Interestingly, the LRP1 levels were higher in mature chondrocytic HCS-2/8 and osteoblastic SaOS-2 than in other cells, whereas the other LDLR family members involved in ossification were detected at lower levels in HCS-2/8. It was interesting to note that in HCS-2/8, LRP1 was observed not only on the cell surface and in the cytoplasm, but also in the nucleus. Exogenously added CCN2 was incorporated into HCS-2/8, which was partially co-localized with LRP1, and targeted to the recycling endosomes and nucleus as well as the lysosomes. These findings suggest specific roles of LRP1 in cartilage biology. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.04.109

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Language：Japanese   Publisher：岡山歯学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Connective tissue growth factor (CTGF/CCN2) can be induced by various forms of stress such as exposure to high glucose, mechanical load, or hypoxia. Here, we investigated the molecular mechanism involved in the induction of ctgf/ccn2 by hypoxia in a human chondrosarcoma cell line, HCS-2/8. Hypoxia increased the ctgf/ccn2 mRNA level by altering the 3'-untranslated region (UTR)-mediated mRNA stability without requiring de novo protein synthesis. After a series of extensive analyses, we eventually found that the cis-repressive element of 84 bases within the 3'-UTR specifically bound to a cytoplasmic/nuclear protein. By conducting a UV crosslinking assay, we found the cytoplasmic/nuclear protein to be a 35 kDa molecule that bound to the cis-element in a hypoxia-inducible manner. These results suggest that a cis-element in the 3'-UTR of ctgf/ccn2 mRNA and trans-factor counterpart(s) play an important role in the post-transcriptional regulation by determining the stability of ctgf/ccn2 mRNA.

DOI： 10.1038/sj.onc.1209129

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

Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)mediated induction of the ctgf/ccn2 gene was a posttranscriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.

DOI： 10.1158/1535-7163.MCT-05-0097

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Communications International Ltd  

Objective: To characterise a natural antisense transcript of connective tissue growth factor. Materials and Methods: RNA from several cell lines was analysed by RNase protection assay to detect antisense transcripts. To characterise the regulatory aspect of the transcribed area, chimeras were constructed in which the firefly luciferase gene was fused with the corresponding segment of ctgf/ccn2, and the gene expression was monitored. Results: A natural antisense transcript complementary to the 3′-untranslated region of ctgf/ccn2 mRNA in cultured human tumour cells was detected. The luciferase gene fused with the full-length antisense 3′-untranslated region showed strikingly low levels of luciferase expression compared with the control. Based on a series of deletion analyses, the major repressive element was located in the middle portion within the 3′-untranslated region, which corresponded to the antisense transcribed area. Conclusion: These results suggest that controlled expression of antisense RNAs against the 3′-untranslated region of ctgf/ccn2 mRNA may be involved in the determination of phenotypes in certain oral malignancies. © 2006 Asian Association of Oral and Maxillofacial Surgeons.

DOI： 10.1016/S0915-6992(06)80014-2

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Background: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro. Results: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium
whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis. Conclusion: These results taken together suggest important roles of CCN2/ CTGF in the development and regeneration of periodontal tissue including the periodontal ligament. © 2005 Asano et al
licensee BioMed Central Ltd.

DOI： 10.1186/1478-811X-3-11

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

The cis-acting element of structure-anchored repression (CAESAR) is a post-transcriptional regulatory element of gene expression, which is located in the 3'-untranslated region (UTR) of the human ccn2 gene (ctgflccn2). In this report, the repression mechanism of CAESAR, as well as the structural requirement, was investigated. Removal of minor stem-loops from CAESAR resulted in proportional attenuation of the repressive function, whereas removal of the single bulge or modification of primary nucleotide sequence did not affect its functionality. In light of functional mechanism, CAESAR exerted no significant effects on stability or nuclear export of the cis-linked mRNA. However, this element significantly interfered with the association of such mRNA on ribosome and slowed down the translation process thereafter in vitro. A translation repression mechanism by RNA secondary structure to determine the basal ctgflccn2 expression level was uncovered herein. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.05.068

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

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo. &COPY; 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2004.10.015

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Background: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. Results: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-α. To induce pro-inflammatory or reparative responses, TGF-β was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-α stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-β and a glucocorticoid. Conclusion: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis. © 2005 Moritani et al
licensee BioMed Central Ltd.

DOI： 10.1186/1478-811X-3-6

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

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.

DOI： 10.1074/jbc.M411632200

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

Previously, we showed that gene expression of the rheurnatoid arthritis-related antigen RA-A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFalpha. Associated with this phenomenon, RA-A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by clownregulation of RA-A47 expression with ra-a47-specific anti-senseoligonucleotide. Here, we show that the enhanced surface expression of RA-A47 on a chondrocytic cell line, HCS-2/ 8 was also a direct result of RA-A47 downregulation by ra-a47 anti-sense oligonucleoticle, independent of the cytokine effects. Moreover, cell-surface expression of CD9, a l integrin-associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra-a47 anti-sense oligonucleoticle-treated cells. The CD9 was colocalized with RA-A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin-V binding to the cell surface and the level of a number of apoptosis-related genes including caspase-9 were increased after ra-a47 anti-sense oligonucleoticle treatment, suggesting that enhanced surface expression of RA-A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metal lothionein-III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti-sense treatment, but not by TNFalpha.. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFalpha. The surface-exposed RA-A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheurnatoid arthritis. J. Cell. Physiol. 202: 191 -204, 2005. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcp.20112

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：岡山歯学会  

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

HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2004.09.039

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.

DOI： 10.1093/jb/mvh126

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BONE & MINERAL RES  

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage.
Introduction: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2.
Materials and Methods: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo.
Results: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which Clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro.
Conclusion: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.

DOI： 10.1359/JBMR.040322

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1. antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.

DOI： 10.1093/jb/mvh042

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

Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor alpha (TNFalpha). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFalpha. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNFalpha, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S-oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNFalpha-independent RA-A47 clownregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFalpha upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNFalpha synthesis. These observations indicate that clownregulation of RA-A47 induces TNFalpha-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10341

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

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

Connective tissue growth factor (CTGF/Hcs24) is a critical growth factor for chondrocytic growth and differentiation. In this report, we describe for the first time glucocorticoid-mediated induction of the CTGF/Hcs24 gene in a chondrocytic cell line, HCS-2/8. Steady-state mRNA levels of CTGF/Hcs24 were remarkably increased after treatment with 50 nM dexamethasone, as confirmed by Northern blotting and quantitative real-time polymerase chain reaction (PCR) analysis. Corresponding to the increase in mRNA, production of CTGF/Hcs24 protein was remarkably enhanced, following a time course of up to 6 h. The observed increase in mRNA can be ascribed to transcriptional enhancement, since the stability of CTGF/Hcs24 mRNA was not affected by the same concentration of dexamethasone, which was indicated by the results of an mRNA degradation assay. However, unexpectedly, the prototypic ctgf/hcs24 promoter was not responsible for the dexamethasone stimulation, suggesting the glucocorticoid receptor binding site(s) to be elsewhere in the CTGF/Hcs24 gene. Enhancement of the prototypic promoter activity by dexamethasone was observed in murine fibroblastic cells, demonstrating the complexity of the regulatory mechanism of ctgf/hcs24 gene expression. Of importance, dexamethasone at the same concentration significantly stimulated proteoglycan synthesis in HCS-2/8 cells up to the same levels as exogenously added CTGF/Hcs24. These findings represent a novel effect of glucocorticoid on the production of CTGF/Hcs24 by chondrocytic cells, and indicate that CTGF/Hcs24 may mediate the stimulative effect of dexamethasone on chondrocytic phenotypes. Also, our results shed light on the complex mechanism of CTGF/Hcs24 induction by glucocorticoids. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S8756-3282(03)00227-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Objective: To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. Design: Five weeks old wild type mate rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. Results: CTGF was expressed strongly in the endothelial. cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important rote in angiogenesis and granulation tissue formation specifically at early heating stage after tooth extraction to initiate alveolar bone repair. Conclusion: CTGF was expressed at early heating stage of the rat tooth extraction wound. (C) 2003 Elsevier Ltd. All. rights reserved.

DOI： 10.1016/S0003-9969(03)00153-5

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Language：English   Publisher：AMER SOC BONE & MINERAL RES  

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

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) plays important roles in the control of the proliferation and differentiation of chondrocytes in vitro. To clarify the mechanisms of regulation by CTGF/Hcs24 with respect to cartilage metabolism, we investigated the interaction between CTGF/Hcs24 and heparan sulfate proteoglycan perlecan. An immunofluorescence study showed that CTGF/Hcs24 was colocalized with heparan sulfate and perlecan in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 in vitro. Northern blot analysis showed that perlecan, syndecan-1, -2, and -4 transcripts were detected in HCS-2/8 cells. Particularly, expression of the perlecan gene increased markedly in HCS-2/8 cells by recombinant CTGF/Hcs24 (rCTGF/Hcs24) treatment. We also found that CTGF/Hcs24 interacted with perlecan from HCS-2/8 cells in vitro. Furthermore, CTGF/Hcs24-stimulated gene expression of the aggrecan gene, as well as DNA/proteoglycan synthesis, was diminished when HCS-2/8 cells were pretreated with heparinase, indicating that the effects of CTGF/Hcs24 on chondrocytes occurred through the interaction between CTGF/Hcs24 and heparan sulfate on the cells. An in vivo study using mouse growth plate revealed that CTGF/Hcs24 produced by hypertrophic chondrocytes was localized from the proliferative to the hypertrophic zone, whereas perlecan was predominantly localized in the prehyphertrophic zone. Consistent with such findings in vivo, the binding of I-125-rCTGF/Hcs24 to maturing chondrocytes was at higher levels than that to chondrocytes in hypertrophic stages. These findings suggest that CTGF/Hcs24 produced in the hypertrophic region may act on chondrocytes in the proliferative and maturative zone via some heparan sulfate proteoglycan, such as perlecan. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jpc.10277

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Language：English   Publisher：ELSEVIER SCIENCE INC  

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

Connective tissue growth factor (CTGF) is known to be a multifunctional growth factor that is overexpressed. in several types of malignancies. In this study, effects of CTGF gene overexpression on the phenotypes of oral squamous cell carcinoma cells were investigated by using a cell line with undetectable endogenous CTGF expression. Surprisingly, our results indicated that CTGF-overexpressed clones were characterized by attenuated cell growth and less potent tumorigenicity, with coincidental downregulation of prothymosin a gene. Although CTGF is known to promote cell proliferation in mesenchymal cells, our present results suggest that CTGF acts as a negative regulator of the cell growth in oral squamous cell carcinoma possibly through its interaction with growth modifiers inside the cell. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(02)00718-8

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Language：English   Publisher：WILEY-LISS  

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10206

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WALTER DE GRUYTER & CO  

CTGF/Hcs24 is a multifunctional growth factor that potentiates the growth and differentiation of various cells. Our previous study revealed that the 3'-UTR of mammalian CTGF/Hcs24 mRNA contains a small segment that represses the gene expression in cis fashion. In this study, we isolated and characterized a chicken CTGF/Hcs24 cDNA clone. Chicken ctgf/hcs24 mRNA showed highly conserved homology in the ORF to that of mammalian species, whereas the homology in the 3'-UTR was relatively low. Northern blotting analysis revealed that chicken ctgf/hcs24 mRNA was expressed most strongly in cartilage, and also in brain, lung, heart, but faintly in liver. Thereafter we analyzed the functional potential of the 3'-UTR of ctgf/hcs24 cDNA to regulate its gene expression by reporter gene assay, and found that it repressed gene expression in cis fashion, specifically in avian cells, but not in mammalian cells. Conversely, the mammalian 3'-UTR showed less repressive activity in avian cells than in mammalian cells. Deletion analysis showed that a segment near the polyadenyl tail of the 3'-UTR of chicken ctgf/hcs24 played an important functional role, unlike in the mammalian species. Thus, we uncovered a novel mode of functional conservation of the ctgf/hcs24 3'-UTR among vertebrate species mediated by different factors.

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

The expression of the connective tissue growth factor (ctgf) gene increases along with the differentiation of growth cartilage cells, and the highest expression is observed in the hypertrophic stage. Similarly, recent reports demonstrated c-fos expression in chondrocytes in the early hypertrophic zone of growth cartilage, and suggested that the c-fos gene may play a crucial role in the regulation of hypertrophic differentiation. A chondrocytic human cell line, HCS-2/8, is known to retain a variety of chondrocytic phenotypes. When such cells were kept overconfluent, they expressed increasing levels of c-fos transcripts along a time course phenotypically similar to that of hypertrophic differentiation. Moreover, by using a competitive electromobility-shift assay, we found that AP-1, a Fos/Jun heterodimer, in HCS-2/8 was capable of binding not only to a typical AP-1-binding DNA fragment but also to the enhancer fragment of the ctgf gene. Based on the findings above, we hypothesize that, prior to hypertrophic differentiation, AP-1-related oncogenes are activated and that their gene products subsequently activate ctgf gene expression, which might eventually induce hypertrophy.

DOI： 10.1007/s00774-003-0410-1

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

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

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：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 &apos;TRENDIC&apos;, 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.

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

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

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：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.

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

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

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：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&apos;-untranslated region (3&apos;-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&apos;-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.

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

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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.

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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.

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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.

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

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

To clarify the multiple functionality of connective tissue growth factor (CTGF), we examined the effects of nascent CTGF within the cell by transient expression. Tn Cos-7 cells, expression of human CTGF induced an altered cell morphology, It was associated with an increased cellular DNA content and loose attachment, indicating the cells mere in G2/M phase. Overexpression of CTGF did not induce cell growth, whereas recombinant CTGF efficiently stimulated the proliferation extracellularly, These results indicate that intracellular CTGF may act as an antimitotic agent, thus it should also be noted that nascent CTGF was found to accumulate around the central mitotic machinery. (C) 2000 Federation of European Biochemical Societies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT MEDICAL PRESS  

Selective cytotoxic effects of gamma-glutamylcysteine ethyl ester (gamma-CCE) against human immunodeficiency virus type 1 (HIV-l)-infected H-9 T lymphocytic cells were demonstrated previously. However, the mechanism of those effects remained unclear. Here, we report on enhanced cytotoxicity of the lentiviral lytic peptide I (LLP-1) of gp41, the envelope transmembrane glycoprotein of HIV-I. in the presence of gamma-GCE. Without gamma-GCE, the cytotoxic effect of LLP-1 was transient, whereas with gamma-GCE, cell death induced by LLP-1 remained continuous until termination. Of note, such effects by gamma-GCE were also observed with another unrelated amphipathic peptide toxin, melittin. These results suggest that the synergistic cytotoxic effect of gamma-GCE and LLP-1 may play a central role in the molecular mechanism of the selective cytotoxicity of gamma-GCE in HIV-l-infected T lymphocytic cells.

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

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it,vas fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR, (C) 1999 Federation of European Biochemical Societies.

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

The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-l cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This configuration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunodeficiency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the first NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three different backbones with RPS25 cDNA fragments. Immunofluorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/nucleolar targeting signal. The identified peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNA binding transactivators.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The anti-human immunodeficiency virus type I (anti-HIV-l) effects of gamma-glutamylcysteine ethyl ester (gamma-GCE; TEI-2306) mere examined in vitro. In initial studies using a vigorously HIV-producing human T-lymphocytic cell line, gamma-GCE displayed a novel biphasic repressive effect on chronic HIV-1 infection that was unlike that of other glutathione prodrugs or other reported antioxidants. In high doses, up to a concentration of 2.5 mM, at which neither glutathione (GSH) nor another GSH precursor has shown inhibitory effects, gamma-GCE potently inhibited the production of HIV-1 by a selective cytopathic effect against infected cells, while the viability and growth of uninfected cells mere unaffected at the same gamma-GCE concentrations. At lower concentrations (200 to 400 mu M), gamma-GCE significantly repressed the virus production from chronically HIV-1-expressing cells without affecting their viability. The discrepancy of the thresholds of the toxic doses between infected and uninfected cells was found to be more than IO-fold. Relatively high doses of gamma-GCE, utilized in acute HIV-1 infection of T-lymphocytic cells, entirely blocked the propagation of HIV-1 and rescued the cells from HIV-1-induced cell death. Furthermore, gamma-GCE at such concentrations was found to directly inhibit the infectivity of HIV-1 within 4 h. Repressive effects of gamma-GCE on acute HIV-I infection in human primary human peripheral blood mononuclear cells were also demonstrated. Here, the anti-HIV-1 strategy utilizing gamma-GCE is removal of both HIV-1-producing cells and free infectious HIV-1 in vitro, in place of specific immunoclearance in vivo, which might lead to an arrest or slowing of viral propagation in HN-l-infected individuals.

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

A peptide signal, which may control nucleo-cytoplasmic protein trafficking, was newly identified in human immunodeficiency virus type I (HIV-1) Rev, a lentiviral post-transcriptional transactivator. The sequence, in the amino-terminal portion of HIV-1 Rev, maintains a Rev mutant with a dysfunctional nuclear/nucleolar targeting signal outside of the nucleus, although this Rev molecule itself is small enough to pass through the nuclear pores. Transition of this sequence to the N-terminus of human T-lymphocytic leukemia/lymphoma virus type I (HTLV-I) p21(x), which is usually located evenly distributed throughout the cell, resulted in capture of p21(x) in the cytoplasm. Mutational analysis clarified that a 14 residue peptide sequence was sufficient to display this inhibitory effect against nuclear entry. Furthermore, this HIV-1 Rev sequence was capable of inhibiting nuclear entry of a fragment of a human ribosomal protein, when it was fused to the carboxy terminus. The identified nuclear entry inhibitory signal (NIS) contains a conserved hydrophilicity motif, which forms an amphipathic helix. Significantly, this motif and its helical structure were shown to be important for NIS function and the HIV-1 Rev function itself. Possible roles for NIS as a molecular anchor are proposed herein.

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

The 5'-RU5 portion of human T-lymphocyte virus type I (HTLV-I) long terminal repeat (LTR) had been reported to contain cis-acting elements for the controlled viral gene expression by the rex gene product. In this study, the human immunodeficiency virus type I (HIV-1) Rev protein was found to enhance gene expression, acting through the 5'-RU5 portion of HTLV-I, while the Rex-responsive element (RxRE)-mediated activation by Rev was reconfirmed to be negative. This positive action of HIV-1 Rev on HTLV-I gene expression seemed to be distinct from the widely accepted Rex or Rev function to facilitate the nuclear export of RxRE-containing unspliced viral mRNAs, since a trans-dominant, nuclear export-deficient mutant (RevM10) still retained the RUB-mediated effector function. Analyses of the functional aspects of Tat/Rev fusion proteins on the HTLV-I RU5 suggested a specific interaction of Rev and RUB, but lacked evidence for the binding of Rev to the RU5 at the RNA level. These results suggest an answer to the controversy regarding a Rex-like function occasionally observed with HIV-1 Rev and its related proteins. It may also be suggested that particular care should be taken when such a trans-dominant Rev mutant is considered to be used as a genetic therapy against HIV-1 infection, in individuals infected with both HIV-1 and HTLV-l. (C) 1998 Academic Press.

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The function of the Rex protein of human T-cell leukemia virus type I (HTLV-1) has been demonstrated to be very similar to the Rev protein of human immunodeficiency virus type 1 (HIV-1). Both of these retroviral regulatory proteins rescue unspliced viral RNAs from the nuclei of infected cells. The Rev protein of HIV-1 has been reported to shuttle between the nucleus/nucleolus and the cytoplasm. Here, we have found that Rex also relocated out of the nucleus in the presence of actinomycin D. This effect was demonstrated in dose- and time-course-dependent manners. In comparison with previous reports on HIV-1 Rev, these effects with Rex seemed to be similar, but less distinct, which may reflect precise differences in the subcellular localization and/or shuttling pathways of Rev and Rex. Interestingly, the endogenous truncated form of the Rex protein, p21(x), significantly interfered with the intracellular translocation of Rex, when coexpressed in trans. As expression of p21(x) occurs in various HTLV-1-infected cells, p21(x) may play a role in the life-cycle of HTLV-1, through regulating the dynamic subcellular distribution of the viral trans-activator, Rex. (C) 1996 Academic Press, Inc.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Intracellular immunization to target the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev has been explored as a genetic therapy for AIDS, Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-I replication have been previously reported by our laboratories, To further understand the molecular mechanism(s) and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D10), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety, These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to alter the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Rev, a major regulatory protein of human immunodeficiency virus type 1, has been demonstrated to shuttle between the nucleus and cytoplasm of infected cells, The fate of the Rev protein in living cells was evaluated by pulse-chase experiments using a transient Rev expression system, Sixteen hours after chasing with unlabelled amino acids, 45% of the labelled Rev was still present, which clearly indicates a long half-life of Rev in living cells, A Rev mutant which is deficient in the ability to migrate from the nucleus to the cytoplasm was degraded more slowly than the wild-type Rev protein, As well, another Rev mutant protein, which lacks a functional nucleolar targeting signal (NOS) and is unable to enter the cell nucleus, was rapidly degraded and undetectable 16 h after chasing, Nuclear-nucleolar targeting properties provided by a divergent NOS from a related retrovirus, which was used to substitute for the NOS of Rev, increased the intracellular half-life of this Rev mutant, Moreover, coexpression of an intracellular anti-Rev single-chain antibody (SFv), which has been shown to interfere with the nuclear translocation of Rev, accelerated the degradation of the wild-type Rev protein, Differential degradation of Rev in the nucleus and cytoplasm may play a critical role in determining and maintaining different stages of human immunodeficiency virus type 1 infection, in conjunction with the shuttling properties of the Rev protein.

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

HTLV-I generally integrates at least one full-length copy in adult T-cell leukemia (ATL) cells, A group of patients without full-length provirus have a unique conserved truncation of the provirus which retains env-pX-3'LTR. Tumor cells of a patient from this group were genetically analyzed, Analysis of the 5' and 3' cellular flanking region adjacent to the provirus suggest that the defective provirus was integrated immediately downstream of a promoter of an unknown cellular gene, The activity of the promoter was weak but was responsive to Tax-like HTLV-I LTR, The provirus may have utilized it as a substitute for the 5'LTR and thus 3'LTR may have become an alternative promoter for the cellular gene, which may give similar viral-cellular interactions to that of general cases with full-length proviruses, Surprisingly, the 3' cellular flanking region which is thought to be controlled originally by the promoter is constitutively expressed specifically in an HTLV-I producing ATL cell line HUT1O2G, in which the corresponding region is not modified by provirus, The detection of this HTLV-I-induced transcript provides a probe to find an HTLV-I inducible unknown cellular gene that may be related to the pathogenesis of ATL.

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

Rex of human T-cell leukemia virus type I (HTLV-I) and Rev of human immunodeficiency virus 1 (HIV-1) are post-transcriptional regulators of viral gene expression. By means of affinity chromatography, we purified an 18-kDa cellular protein that bound to the conserved leucine-motif/activation domain of HTLV-I Rex or HIV-1 Rev. The protein that was purified through a Rev-affinity column was found to bind to Rex immunoprecipitated with anti-Rex IgG from an HTLV-I-producing cell line. We analyzed the purified approximate to 18-kDa protein biochemically and identified it as prothymosin alpha. The binding activity of prothymosin alpha to Rev or Rex was completely abolished when the epsilon-amino groups of its lysine residues were chemically modified by N-succinimidyl-3-(4-hydroxy-3,5-diodo-phenyl)propionate. The functional relationship between the nuclear protein prothymosin alpha and Rex-Rev is discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Applications of transdominant mutants of human immunodeficiency virus type 1 (HIV-1) regulatory proteins, especially Rev mutant, have been attempted for gene therapy against AIDS, because the Rev protein is essential for viral replication. We have previously reported that a mutant Rev protein (dRev) lacking its nucleolar targeting signal remained out of nuclei in expressed cells and strongly inhibited the function of Rev. To investigate the effects of dRev on HIV-1 replication, we established several dRev-expressing human cell lines with two different vector systems and examined virus production in these cells, An HIV-1-derived vector containing drev cDNA was constructed and introduced into CD4-positive HeLa cells and cells of the human T-cell line CCRF-CEM (CEM). In dRev-expressing HeLa cells, virus replication, syncytium formation, and cell death caused by HIV-1 infection were remarkably suppressed, and the same vector also conferred a resistant phenotype on CEM cells, The production was also suppressed in CEM cells containing the drev gene driven by a cytomegalovirus promoter. In addition, we found that dRev dia not cause nucleolar dysfunction in a transient assay, in contrast to other transdominant mutants and wild-type Rev, Since dRev cannot migrate into the nuclei, it is expected not to interfere with nuclear/nucleolar functions of the host cell, We conclude that dRev is one promising candidate as an antiviral molecule for gene therapy against AIDS,

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

The Rev protein of human immunodeficiency virus type-1 is an RNA-binding posttranscriptional transregulator encoded by an accessory gene that is distinct from retroviral oncogenes and whose origin is unclear. We hypothesize that the a ev gene was generated by duplication of a viral RNA segment having a secondary-structure that evolved into the Rev-responsive element (RRE). This hypothesis is based on the following findings. First, accumulated data on functional mapping of Rev, Tat, and the transmembrane protein of Env suggested that the major coding exon of rev should have been inserted into the transmembrane region of env during the course of its evolution. Experiments with equine infectious anemia virus, another complex retrovirus, also indicate that a large portion of rev is located within the dispensable transmembrane region of env. Second, base usage analysis suggests the same origin for rev and RRE. Our hypothesis may provide a new insight into the evolutionary aspect of RNA-binding transactivators.

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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

Retroviruses generally integrate as proviruses which are flanked by long-terminal repeats (LTRs) on both 5' and 3' ends. Since these LTRs are required for the efficient integration mediated by the viral integrase, it is believed that defective proviruses with a single LTR are normally formed by deletion after integration. However, we found no deletion of cellular sequences around the integration site of such a defective HTLV-1. Rather, we identified 99 bp-long direct repeats adjacent to both ends of the defective provirus. The repeated cellular sequences contained a potential poly(A) signal followed by a retroviral primer-binding-site-like sequence. The presence of the direct repeats of cellular sequences can be explained by the integration of the defective virus through homologous recombination between cellular and viral read-through sequences.

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

A nonfunctional mutant of human immunodeficiency virus type 1 Rev was created by deleting seven amino acid residues within the nucleolar targeting signal. This mutant Rev remained in the cytoplasm in expressed cells and strongly inhibited the function of Rev by interfering with the nuclear/nucleolar localization of coexpressed Rev. These findings strongly suggest the multimerization of Rev in the cytoplasm before migration to the nucleus/nucleolus, where wild-type Rev functions as a trans-regulator.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

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The posttranscriptional regulator (rex) of human T-cell leukemia virus type I is known to be located predominantly in the cell nucleolus and to induce the accumulation of gag and env viral mRNAs. The N-terminal 19 amino acids of rex-encoded protein (Rex) has been shown to be sufficient to direct hybrid proteins to the cell nucleolus. We have studied the function of the nucleolar targeting signal (NOS) of rex by using full-length proviral DNA and mutant rex expression plasmids. Partial deletions of the NOS sequence abolished the accumulation of unspliced cytoplasmic mRNA, although the gene products of rex mutants were found in the nucleoplasm. These results indicate that NOS sequence, or nucleolar localization of Rex, is essential for Rex function.

DOI： 10.1073/pnas.86.24.9798

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

The trans-acting factor of human immunodeficiency virus (HIV), Tat, has a basic amino-acid cluster that is highly conserved among different HIV isolates. We have examined the effects of mutations in the basic region of Tat on its trans-acting activity and cellular localization. Introduction of a stop codon immediately preceding the basic region abolished the activity, while the truncated mutant with the basic region retained some activity. The basic region of Tat was replaceable with that of Rev (another trans-acting factor of HIV) but not with that of adenovirus Ela nor cellular enzyme. The result of immunofluorescence analysis revealed a correlation between the nuclear, especially nucleolar, accumulation and the activities of mutant Tat proteins.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本整形外科学会  

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Language：English   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publisher：(一社)日本生理学会  

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：English   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：岡山歯学会  

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