Fourth international workshop on the CCN family of genes: Abstracts and posters October 18–21, 2006

Regulation of the CCN2 gene by mechanical forces in smooth muscle cells

Brahim Chaqour, Ru Yang, Quan Sha

SUNY Downstate Medical Center

The CCN2 gene is strongly induced in smooth muscle-rich hollow organs which experience mechanical overload arising from either hypertension, altered hemodynamics or obstruction. This suggests that mechanical forces might be the primary inducers of the CCN2 gene in smooth muscle cells. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro, and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. Molecular analyses of the CCN2 promoter revealed that a highly conserved NF-κB binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-κB binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, inhibition of either actin polymerization or actin signaling-dependent pathways significantly altered the CCN2 promoter activity. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle alpha-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-1B and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-κB activation and induce CCN2 gene expression.

The 3′-untranslated region-mediated regulation of the ccn family genes

Satoshi Kubota¹, Yoshiki Mukudai², Harumi Kawaki¹, Masanao Minato¹, Takuo Fujisawa¹, Yosuke Nakagawa¹, Machiko Honda¹, Seiji Kondo¹, Masaharu Takigawa¹

¹Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and ²Biomedical Research Center, Okayama University Dental School

The 3′-untranslated region (UTR) of the mRNA has been recently attracting the interest of a number of molecular biologists, since it contains a variety of post-transcriptional regulatory elements of gene expression.

Over the years, we have characterized the regulatory function of the 3′-UTRs of the ccn family genes. In the 3′-UTR of the best-characterized member, ccn2, we already identified a few post-transcriptional RNA elements that are critically involved in the regulated ccn2 expression in chondrocytes during differentiation. Subsequently, we have been characterizing the 3′-UTRs of the other members as well. Interestingly, recent evaluation indicates that the 3′-UTRs of human ccn1, ccn3, ccn4 and ccn5 are also repressors of gene expression. Among these 4, the 3′-UTR of ccn4 has been shown to be involved in the gene induction that is specific in osteogenic cells. Namely, the repressive effect of the 3′-UTR is attenuated in human chondrocytic and osteoblastic cells, which is consistent with the prolonged intracellular half life and higher steady-state level of the ccn4 mRNA in those cells. The critical involvement of the 3′-UTRs in the gene regulation of most members suggests the 3′-UTR as a fundamental constituent in the ccn family as a part of regulatory machinery.

Expression patterns and phenotypic correlations of CCN1-3 in an organotypic model of neurogenesis

Herman Yeger¹, Micky Tsui¹, Bernard Perbal²

¹The Hospital for Sick Children and ²Université Paris 7 D. Diderot

Expression profiles of the structurally related CCN 1–3 proteins in normal tissues, tumors and cell lines have indicated differential and dichotomous patterns suggesting functional complexity. In order to recapitulate and study complex tissue organization ex-vivo we are exploring a variety of cell substrata that mimic the 3D extracellular matrix. The multipotential NT2 teratocarcinoma model of neurogenesis can be induced to undergo CNS tissue specific differentiation in response to inducers like retinoids. NT2 cells were seeded on UltraWeb, a 3D polyamide synthetic fiber substratum mimicking extracellular matrix, induced with retinoic acid (RA), and differentiation followed over time. These cultures were compared to traditional RA induced NT2 embryoid bodies plated on a polylysine/laminin substratum and to uninduced controls. Cultures were immunophenotyped for expression of CCN 1–3, markers of neuronal, glial and epithelial cell types, and proliferation (BrdU labeling, Ki–67). UltraWeb cultures showed a remarkable higher degree of neuronal differentiation and maturation with extensive axonal and dendritic outgrowths. Developing neurons strongly up-regulated expression of CCN3 correlating with maturation. Mesenchymal phenotypes showed expression of CCN2. The tissue level organization complexity of UltraWeb cultures provides an excellent model to investigate CCN expression and means to manipulate phenotypes with biochemical and molecular tools.

CCN2 (connective tissue growth factor) supports the binding of fibronectin to alpha5beta1 integrin and maintains integrin signaling

Takashi Nishida¹, R. Andrea Deyoung², Harumi Kawaki¹, Masaharu Takigawa¹, Karen M. Lyons²

¹Department of Biochemistry and Molecular Dentistry, Okayama University of Medicine, Dentistry and Pharmaceutical Sciences and ²Department of Orthopaedic Surgery, Department of Molecular, Cell, and Developmental Biology, David Geffen School of Medicine at UCLA

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is thought to be the major mediator of excess extracellular matrix (ECM) accumulation in most fibrotic diseases. A role for CCN2 as an essential mediator of ECM composition was revealed through analysis of ccn2 deficient mice. These mice die at birth due to complications resulting from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using chondrocytes isolated from ccn2-deficient mice. Expression of collagen type II and aggrecan were decreased in ccn2 mutant chondrocytes, confirming a defect in ECM production. ccn2-/-chondrocytes also exhibited impaired DNA synthesis, reduced cartilage ECM synthesis, and impaired adhesion to fibronectin. This latter defect is associated with decreased expression of α5 integrin. Moreover, CCN2 acts directly as a ligand for α5β1 integrin in chondrocytes. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in mutant cells. These findings argue that CCN2 exerts its major effects in chondrocytes due to its ability to engage integrins and activate integrin-mediated signaling pathways.

Identification of CCN2/CTGF binding proteins from human chondrocytes

Takako Hattori¹, Daisuke Araki¹, Mitsuhiro Hoshijima¹, Eriko Aoyama¹, Miho Inoue¹, Hiroshi Hanagata², Akira Miyauchi³, Masaharu Takigawa¹

¹Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, ²R & D Department, Higeta Shoyu Co. Ltd., Choshi, Japan, and ³Choshi Laboratory, ProteinExpress Co. Ltd., Choshi, Japan

To identify CCN2/CTGF interactive proteins which modulate its action, we carried out a yeast-two hybrid screening using CCN2 as bait and a cDNA library from the chondrocytic cell line HCS-2/8, and we found two CCN2-interacting proteins, fibronectin 1 and matrilin 3. For direct binding to fibronectin1, only the C-terminal domain (CT) of CCN2 was enough; the binding enhanced adhesion of HCS-2/8 cells to fibronectin 1 through the integrin receptor α5β1, but did not involve αvβ3 as shown by blocking with specific antibodies against integrins. Matrilin-3 is a non-collagenous extracellular matrix protein, which associates with collagen and plays a role in collagen fibril assembly. We demonstrated that CCN2 binds to matrilin-3 in a dose-dependent manner, and colocalizes with matrilin-3 outside of the cells. Matrilin-3 supported HCS-2/8 adhesion in a dose dependent manner and CCN2 enhanced HCS-2/8 adhesion to matrilin-3. An anti-matrilin-3 antibody which inhibits adhesion of HCS-2/8 cells to matrilin-3 did, however, not inhibit CCN2-enhanced adhesion to matrilin-3. These results suggest that CCN2 is not only a growth factor, but also a factor which supports multiple specific interactions with proteins of the extracellular matrix, cell-matrix interactions, and the assembly of extracellular matrices.

Notch signal and CCN3 in the bone marrow derived mesenchymal stem cells

Ken-ichi Katsube

Tokyo Medical and Dental University

Regulation of bone marrow stem cells (BMSC) seems rather different from authentic stem cells. To investigate their property, we focused on Notch signal, one of the principal systems of stem cells. Notch signal stimulates the neurogenesis and suppresses the osteogenesis in the BMSC whereas it suppresses the neurogenesis of neural stem cells and stimulates the osteogenesis of osteoblasts. One of the BMSC, called Kusa was chosen for its analyses from its multipotential property. We examined the downstream of Notch signal in Kusa by gene chip and found the CCN3 expression dynamically changed depending on the level of the Notch signal. Previously we reported the Notch signal activation by CCN3, which implies a positive feedback between them. Several truncated forms of CCN3 were introduced expecting a dominant negative or constitutive active effect. A truncated form of CT domain upregulated both neurogenesis and osteogenesis of Kusa. We again performed a gene chip to this transformant and found that several bHLH transcrition factors are upregulated. Authentic Notch downstream such as Hes family is moderately upregulated by Notch in Kusa, but other bHLH is upregulated in this transformant. We are evaluating the mutual effect of these bHLH factors from the aspect of CCN3/Notch.

Antiproliferative activity of CCN3-qualitative and quantitative aspects

Bernard Perbal, Anne Marie Bleau, Noureddine Lazar, Nathalie Planque

Laboratoire d’Oncologie Virale et Moléculaire, Université Paris 7, D. Diderot Paris, France

Originally discovered as an integration site of Myeloblastosis Associated Virus [MAV-1(N)] in avian nephroblastomas, the ccn3 gene was previously reported to encode a protein with potential tumor-suppressor activity. In avian nephroblastomas, an elevated expression of ccn3 has been observed, as compared to the levels of ccn3 expression in normal kidney. We have shown that the increased expression of ccn3 in these tumors is related to the differentiated status of the tumor cells, and established that MAV target cells are blastemal cells commited to epithelial differentiation. In normal conditions, the expression of ccn3 is submitted to a tight spatio temporal regulation and is associated to the differentiation of several tissues including kidney, bone, and muscle.

In nephroblastoma, proviral MAV integration was reported to occur within the ccn3 coding sequences and resulted in the high expression of an aminotruncated CCN3 protein showing transforming properties when assayed on normal chicken embryo fibroblasts. On the contrary, the full length CCN3 protein showed an antiproliferative activity on the same cells. In order to determine whether the expression of CCN3 was impaired in human tumors, we have analyzed several different types of tumor cells for their content and expression of CCN3. We have also used a series of recombinant plasmids that allow the expression of CCN3 in transfected cells and tested the ability of siRNAs to suppress the biological properties of CCN3. In all the cases that we have studied, the expression of a full length CCN3 protein suppressed tumor cell growth. Furthermore, similar results were obtained with normal cells, indicating that the negative activity of CCN3 on cell growth was not restricted to abnormal situations. The antiproliferative activity of the CCN3 protein was also demonstrated by the use of exogenous recombinant proteins and specific antibodies. Interestingly, in some tumor cells, the elevated expression of CCN3 was associated to a significant higher risk of developing metastasis and increased migratory capacity.

In this communication we will present recent results that highlight potentials mechanisms that support the negative role of CCN3 in the regulation of cell proliferation.

Interaction of gap junction protein connexin 43 and CCN3 in breast cancer cells

Wun Chey Sin¹, Nathalie Planque², Bernard Perbal², Christian C. Naus¹

¹University of British Columbia and ²Université Paris 7, D. Diderot

We have previously shown that overexpression of gap junction protein Cx43 reverses the oncogenicity of glioma cells and that enhanced expression of Cx43 facilitates the redistribution of CCN3 to gap junction plaques containing Cx43, as well as the secretion of CCN3. We next determine the role of Cx43 and CCN3 in affecting the oncogenicity of breast cancer cells. We achieved a reduction of Cx43 in Hs578T cells by using Cx43 siRNA that resulted in the translocation of CCN3 from the cytoplasm into the nucleus. In addition, we observed a decrease in CCN3 expression in Hs578T-Cx43siRNA cells compared to Hs578T cells transfected with the control vector (Hs578T-control). To determine whether the C-terminal tail of Cx43 is necessary for CCN3 translocation in breast cancer cells, we overexpressed Cx43t, a truncated mutant of Cx43 lacking the C-terminal tail known to interact with CCN3, in Hs578T cells. Hs578T-Cx43t exhibited a strong nuclear localization of CCN3. In contrast, overexpression of full length Cx43 in Hs578T cells (Hs578T-Cx43) results in the absence of CCN3 from the nucleus. Taken together, these preliminary findings suggest Cx43 is necessary for both the upregulation and subcellular localization of CCN3 in breast cancer cells.

Gap junction protein connexin 43 upregulates CCN1 in C6 glioma cells

Wun Chey Sin¹, Dave C. Bates¹, John F. Bechberger¹, Walter J. Rushlow², Christian C. Naus¹

¹University of British Columbia and ²University of Western Ontario

Gap junctions are composed of transmembrane proteins called connexins. They form channels to allow exchange of materials between cells and are believed to mediate growth control by permitting intercellular communication to occur. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes. As gliomas are transformed astrocytes with reduced Cx43 expression, we seek to identify genes that are regulated by Cx43. Here, we report the overexpression of Cx43 in C6 glioma cells (C6-Cx43) upregulates the expression of CCN1/Cyr61. Serum stimulation of C6-Cx43, but not parental C6 cells, induced the expression of CCN1 protein and this enhancement was maintained for a further 6 hours. We have previously shown that CCN3/NOV is associated with Cx43 at the gap junction plaques. In contrast, CCN1 has a diffuse cytoplasmic staining without apparent co-localization with Cx43. Overexpression of CCN1 in C6 glioma cells increased growth rate of glioma cells under serum-limiting condition. In contrast, overexpression of Cx43 in glioma cells reduced growth rate. The dissimilar cellular properties of CCN1 and Cx43 overexpressors indicate that CCN1 is not the major mediator of Cx43-dependent growth control.

CTGF/CCN2 inhibits adipocyte differentiation

Stephen M. Twigg, Susan V. McLennan, Paul F. Williams, Joanne T.M. Tan

Discipline of Medicine, The University of Sydney and Royal Prince Alfred Hospital, Sydney, Australia

Fat cell differentiation (FCD), or adipogenesis, is impaired in the metabolic syndrome. TGF beta is known to inhibit FCD and as CTGF/CCN2 may mediate TGF beta bioactivity, we determined if CTGF regulates FCD. The 3T3L1 cell line was differentiated in vitro by IBMX, dexamethasone and insulin. FCD at d10 was confirmed by measures of key mRNA markers, PPAR gamma and adiponectin, and also by triglyceride accumulation (Oil red O) and induced lipogenic enzyme activity, G3PDH. In parallel wells, cells were treated repeatedly each 2d of FCD, or by a single treatment on d5, 8 or 10, with either rhCTGF (250 ng/ml) or active rhTGF beta1 (2 ng/ml). Across 3 independent experiments, both CTGF and TGF beta1 were found to prevent FCD with ∼30–50% inhibition of induced PPAR gamma and adiponectin mRNA (each P < 0.05 by ANOVA with repeated measures), and reduced lipid accumulation. We have recently shown that CTGF is increased in central adipose depots in fat fed mice compared with subcutaneous sites and also with adipose depots in standard chow fed animals, further suggesting that CTGF may impair adipogenesis in vivo. This data collectively indicates that CTGF/CCN2 inhibits fat cell differentiation. The cellular mechanism of this effect is being explored.

Supported by University of Sydney Sesqui grant.

CCN5 suppresses restenosis in a mouse vascular injury model

Jennifer Malmquist¹, Mark Aronovitz², Michael Mendelsohn², Richard Karas², John Castellot¹

¹Department of Anatomy and Cell Biology, Tufts University School of Medicine, and ²Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston MA, USA

CCN5 is a growth arrest-specific gene in vascular smooth muscle cells (VSMC). In culture, growth-arrested VSMC strongly express CCN5; expression levels decrease during exponential growth. CCN5 overexpression inhibits VSMC proliferation, motility, and invasiveness, while knockdown via siRNA has opposite effects. In vivo, CCN5 expression dramatically decreases in proliferating VSMCs in a rat carotid artery balloon vascular injury model. To evaluate the role of CCN5 in VSMC proliferation in vivo, we examined its temporospatial expression pattern following a carotid ligation injury in mice. We measured medial thickening and neointimal area to evaluate VSMC hyperplasia. Mice were also treated with BrdU to determine cell cycle status. CCN5 protein expression in injured arteries decreases dramatically in the media and newly forming neointima, but increases in the neointima as VSMC proliferation decreases. We also examined whether CCN5 overexpression via an adenoviral vector (AdCCN5) protects wild-type mice from neointimal hyperplasia after carotid ligation injury. AdCCN5 infection strongly suppressed neointimal formation at the approximate midpoint of the injured section of vessel, while significant neointima was present in animals infected with control adenovirus. These data warrant further testing of the therapeutic potential of CCN5 following vascular surgery.

NIH Grant HL49973

Roles of CCN proteins in skeletal growth and regeneration

Masaharu Takigawa¹, Takuo Fujisawa^1,2, Gen Yoshimichi¹, Harumi Kawaki¹, Kazumi Kawata¹, Takeshi Yanagita¹, Seiji Kondo¹, Yoshiki Mukudai¹, Akiko Suzuki³, Takako Hattori¹, Takeyasu Maeda³, Satoshi Kubota¹, Takuo Kuboki², Bernard Perbal⁴, Karen M. Lyons⁵

¹Department of Biochemistry and Molecular Dentistry and ²Department of Oral and Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine and Dentistry, Okayama, ³Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, ⁴Laboratoire d’Oncologie Virale et Moléculaire, UFR de Biochimie, Université Paris, France, ⁵Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

We have shown that CCN2 plays important roles in skeletal formation and regeneration including endochondral ossification. Here we report on new findings in this subject and the roles of other CCN proteins in chondrocyte differentiation during endochondral ossification.

CCN2 promoted the proliferation and expression of the differentiated phenotype of auricular chondrocytes in vitro and cartilage formation in vivo, suggesting that CCN2 plays important role in development and regeneration of elastic cartilage, such as auricular cartilage.

PKC was an upstream driver of multiple signal-transducing kinases including ERK and p38 MAPK that mediated chondrocyte proliferation and differentiation, respectively. JNK also mediated CCN2 signals independently. Moreover, PI3K transduced CCN2 signals to promote chondrocyte hypertrophy.

Both in wild-type and the CCN2-mutant mice, the five CCN members were differentially produced depending upon chondrocytic differentiation stages. CCN2 deletion upregulated CCN3 and downregulated CCN6 in cultured chondrocytes. CCN4 and its variant mRNAs were detected in growth plate chondrocytes, especially hypertrophic chondrocytes. Overexpression of CCN4v in chondrocytic cells enhanced hypertrophic phenotype. These findings suggest that not only CCN2 but also CCN3, 4 and 6 are involved in terminal differentiation of growth plate chondrocytes.

Concerning CCN2 signal transduction in cartilage, we will also report possible role of LRP1.

CCN1 and CCN2 are required for skeletal and vascular development

Karen Lyons¹, Takashi Nishida², R. Andrea DeYoung¹, Ruth Baxter¹, Lisa Dornbach¹, Marcos Pacheco³

¹University of California, 2641 MacDonald Research Laboratory and ²Okayama University, Okayama, Japan, ³Universidade Federal do Rio de Janeiro

CCN1/Cyr61 and CCN2/Connective Tissue Growth Factor are structurally related matricellular proteins with diverse activities. However, little is known about their roles during development. We characterized mice carrying null or conditional floxed alleles of ccn1 and ccn2 to address this issue.

Both CCN1 and CCN2 are essential for chondrogenesis, but have distinct roles. CCN1 is required for joint formation, whereas CCN2 is essential for formation of the growth plate. These divergent functions are related in part to distinct patterns of expression. The effects of CCN2 may be mediated through effects on expression of multiple integrins. In addition, CCN2 is a ligand for integrins in chondrocytes. In accordance, ccn2-/-chondrocytes exhibit defects in adhesive signaling. Analysis of Ccn2 mutants reveals a role for CCN2 in angiogenesis. Ccn2 mutants exhibit edema, elevated levels of VEGF, and defects in sprouting angiogenesis. Moreover, Ccn2fx/fx;VE-CAD mice, lacking CCN2 in developing vasculature, exhibit early postnatal lethality.

Finally, we examined dermal development in ccn2 mutants. This revealed that the dermal microvasculature is defective, and that dermal fibroblasts are unable to undergo contraction in response to TGF beta.

These studies reveal both overlapping and distinct functions for CCN1 and CCN2 in angiogenesis, dermal development, and skeletogenesis.

Mechanical stress-related action of NOV (CCN3) together with periostin in the periodontal ligament and periosteum

Akira Kudo

Department of Biological Information, Tokyo Institute of Technology

Periostin is expressed in the periodontal ligament and periosteum. We generated the periostin deficient-/-mice, and revealed the disappearance of the shear zone in the periodontal ligament because of a failure in digestion of collagen fibers, suggesting that periostin functions in the remodeling of collagen matrix in the periodontal ligament. In the periosteum, mechanical loadings were applied periodically to mouse tibia. The results showed no observation of an ectopic bone formation in the periostin-/-mice, indicating that periostin is essential for mechanical stress-dependent bone formation in the periosteum. In the molecular analyses, periostin was associated with the EGF repeats of Notch1, and it enhanced the Hes1 promoter activity. Furthermore, periostin associated with CCN3, and this binding site was different from that of between Notch1 and periositn or CCN3. We also showed that the interaction of periostin and CCN3 enhanced the Notch signaling activity, up to 10 fold, in a cooperative manner. Periostin was co-localized with Notch1 or CCN3 in the periodontal ligament and periosteum, however, in the periostin-/-mice, Notch1 and CCN3 stayed in a cell, suggesting that periostin together with CCN3 functions in transportation of Notch1 from cytoplasm to cell membrane in the process of Notch1 activation.

Alveolar bone regeneration using human bone marrow stromal cells and CCN2

Takuo Kuboki¹, Mitsuaki Ono¹, Satoshi Kubota², Takuo Fujisawa¹, Manabu Kanyama¹, Wataru Sonoyama¹, Harumi Kawaki², Kentaro Akiyama¹, Masamitsu Oshima¹, Nawachi Kumiko¹, Takashi Nishida², Masaharu Takigawa²

¹Department of Oral and Maxillofacial Rehabilitation and ²Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Oral implant therapy is a promising prosthetic solutions for tooth edentulism. However, without enough bone mass, the modality cannot guarantee its long-term stability and function. To solve this problem, auto-transplantation of bone marrow stromal cells (BMSCs), have been studied, without stable clinical outcome. In the previous study, we found that CCN2 was expressed strongly in the endothelial cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. Thus, CCN2 may play an important role in initiating alveolar bone repair. In this study, effects of CCN2 on human BMSCs were examined. To evaluate the effect of CCN2 on cell attachment, we adsorbed CCN2 onto plastic or hydroxyapatite (HA) plates, and then seeded them with hBMSCs. CCN2-mediated migration in vitro was evaluated by use of a modified Boyden chamber method. For estimating the effect of CCN2 in vivo, we cultured hBMSCs cultured on porous HA carriers, and then transplanted them subcutaneously into immunocompromised mice. Consequently, CCN2 significantly enhanced the hBMSC attachment, and this enhancement was negated by the anti-integrin α_Vβ₃ antibody. The migration of hBMSC was also increased dramatically by CCN2. Moreover, CCN2 accelerated the hBMSC migration and survival inside the porous HA in vivo. These results strongly suggest the utility of CCN2 in alveolar bone regeneration in combination with hBMSCs and an HA scaffold.

Expression, regulation and function of CCN2 during odontogenesis

Manabu Kanyama^{1, 2}, Hiroki Sugito¹, Takuo Kuboki², Maurizio Pacifici¹, Eiki Koyama¹

¹Orthopaedic Surgery, Thomas Jefferson University Medical School, Philadelphia PA, USA ²Oral and Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

CCN2 is a potent cytokine which has important roles in cell differentiation and is believed to mediate epithelial-mesenchymal interactions in developing organs. Given that odontogenesis involves similar epithelial–mesenchymal interactions and cytodifferentiation processes, we asked whether CCN2 is expressed during mouse tooth development, how the expression is regulated, and what roles it may have. In situ hybridization revealed that CCN2 was present in epithelial and mesenchymal cells at developmental periods including bud stage, enamel knot and differentiating preameloblasts at cap/bell stages; however, the expression was down-regulated in enamel secreting ameloblasts. Since these expression patterns were similar to those of BMP-4, we asked whether BMP-4 may regulate the expression of CCN2 and found that BMP-4 induced CCN2 in dental mesenchyme, while BMP antagonist, Noggin, blocked CCN2 expression. The CCN2 expression started after 1 hr of BMP-4 treatment and disappeared by 48 hrs. To see the roles in early odontogenesis, cultured mandibular mesenchymal cells were treated with recombinant CCN2. rCCN2 stimulated proliferation of the mesenchymal cells in a dose dependent manner. Taken together, our data demonstrate that CCN2 expression is confined to specific sites and times in the developing tooth germ, is regulated by BMP-4, and appears to function as a mitogen for early odontogenic mesenchyme.

Expression of CTGF mrna and induction of apoptosis in osteocytes by mechanical stimulation in mice

Teruko Takano-Yamamoto¹, Yuichi Sakai², Shingo Kuroda², Tomohiro Fukunaga², Nagato Tamamura², Masaharu Takigawa³

¹Division of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, Tohoku, ²Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, ³Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

In the present study, using a mouse experimental tooth movement model, we investigated the involvement of CTGF in apoptosis of osteocytes during alveolar bone remodeling stimulated by mechanical force. The number of CTGF mRNA-expressing osteocytes increased maximally at 12 hours from the initiation of tooth movement, and decreased with time from days 1 to 21. Osteocytes positive on TUNEL staining, which identifies DNA fragmentation, increased on the pressure side of the alveolar bone at 12 hours after the initiation of mechanical stimulation, and then decreased with time from days 1 to 21. At 12 hours of mechanical stimulation, many ISOL staining-positive osteocytes were noted on the pressure side of the alveolar bone, suggesting that the death of osteocytes was due to apoptosis. On the tension side of the alveolar bone, no change was noted in the CTGF mRNA expression level, nor were there increases in the apoptosis-positive reaction and the number of acellular bone cavities. In addition, the number of acellular bone cavities markedly increased on day 3 of mechanical stimulation then decreased with time from days 7 to 21. After that, the number of osteoclasts markedly increased on the pressure side of the alveolar bone on day 7. These findings suggested that CTGF expressing osteocytes might play a significant role for triggering bone remodeling and induce osteocyte apoptosis in the response of bone to mechanical stimulation.

Involvement of CCN1:α _v β ₃ and ccn1:α ₆ β ₁ integrin binding in acquisition of antiestrogen resistance and tumor progression

Ruth Lupu¹, Lester Lau², Hong Liu¹, Ingrid Espinoza¹

¹Evanston Northwestern Healthcare Research Institute, ²University of Illinois Chicago, Northwestern University Feinberg School of Medicine, IL

The pro-angiogenic regulator CCN1 is overexpressed in invasive breast cancer cells and tumor biopsies. The expression of CCN1 is sufficient to promote breast cancer cell proliferation, cell survival and chemosensitivity through a α _v β ₃ integrin-activated ERK1/ERK2 MAPK signaling. Additionally, CCN1 induces breast cancer cells to bypass the normal estrogen requirements for growth and acquire an estrogen-independent and antiestrogen resistant phenotype. This is apparently mediated via the interaction with the integrin receptor α _v β ₃. Here, we investigated whether expression of CCN1 mutants with substitutions at the α _v β ₃ (D125A) or α ₆ β ₁ (TM) binding domains might impede CCN1’s ability to induce an estrogen (E2)-independent and/or the antiestrogen resistant phenotype in MCF-7 cells. To do so, retroviral infection was used to generate MCF-7 stably cell lines expressing either the CCN1-D125A or the CCN1-TM cDNA variants. Matched controls expressing an empty pBabe vector were also generated. CCN1 expression levels were monitored by immunoblotting. To determine the response of the MCF-7/D125 and MCF-7/TM cells to estrogen and antiestrogen stimuli we performed a series of studies in the presence or absence of E2 and in the presence or absence of the antiestrogens Tamoxifen and ICI 182,782. Our data demonstrated that mutation of CCN1 at the α _v β ₃ and α ₆ β ₁ integrin sites impairs CCN1-induced cell proliferation; the α ₆ β ₁-binding domain is critical for the CCN1-triggered signaling that results in a hormone-independent phenotype in breast cancer cells. Moreover our data demonstrates a differential role for the CCN1 integrin binding domains in the regulation of the cell proliferation and hormone response in breast cancer. These results reveal that CCN1 signaling through α ₆ β ₁ integrin allows the acquisition of an E2-independent and antiestrogen resistant phenotype in breast cancer cells. Moreover, cell cycle progression analysis of MCF-7/D125A and MCF-7/TM cells showed a decreased proliferative response in comparison to the MCF-7/CCN1 cells as measure by flow cytometric analysis. Collectively, these data point toward a differential role for the CCN1 integrin binding domains in the regulation of the cell proliferation and hormone response in breast cancer.

Functional roles of CCN3 in chronic myeloid leukaemia

Sandra Irvine¹, Wanhua Lu¹, Susan Price¹, Nathalie Planque², Nourredine Lazar², Bernard Perbal², Anthony Whetton³, Lynn Mc Callum¹

¹Queen’s University Belfast, Dept. Haematology Research and ²Laboratoire d’Oncologie Virale et Moléculaire, Université Paris 7, D. Diderot, Paris, France, ³Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. We have previously identified downregulation of CCN3 as a consequence of BCR-ABL kinase activity in human CML cell lines and primary human cells. This study investigates the functional consequence of expressing CCN3 in BCR-ABL + cells.

CCN3 function was investigated in K562 cells transfected with vector alone or vector containing a full-length CCN3 construct using flow cytometry and colony formation on methyl cellulose. CCN3 expression resulted in an accumulation of cells in the subG₀ phase of cell cycle (mean for subG₀ 9.9%±4.6 and 21.8%±0.7 for vector alone and vector containing CCN3 respectively; n = 3, p = 0.028). CCN3 expression significantly reduced the clonogenic capacity of BCR-ABL + cells on methyl cellulose by one third in comparison to cells transfected with vector alone (n = 3, p = 0.027). Cells were then treated with recombinant CCN3 which showed a significant increase in the number of cells in the subG₀ area (9.3%±3.9 to 23.7%±6.9 for control and treated cells respectively; n = 3, p = 0.014).

This study demonstrates CCN3 expression in BCR-ABL + cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.

CCN3 expression is reduced as a result of bcr-abl kinase in chronic myeloid leukaemia

Lynn McCallum¹, Wanhua Lu¹, Susan Price¹, Rachel Frazer¹, Nathalie Planque², Bernard Perbal², Anthony Whetton³, Alexandra Irvine¹

¹Queens University Belfast, Dept. Haematology Research and ²Laboratoire d’Oncologie Virale et Moléculaire, Université Paris 7, D. Diderot, Paris, France, ³Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. This kinase is associated with downregulation of CCN3 in human CML cell lines and primary human cells. The aim of this study was to investigate the relationship between BCR-ABL and CCN3 expression.

Real-time PCR was used to detect BCR-ABL and CCN3 expression. The CML cell line, K562, showed high BCR-ABL expression whilst CCN3 was not detected. Treatment with siRNA directed against BCR-ABL resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (n = 3, p = 0.001); treatment with the Bcr-Abl inhibitor, imatinib, showed a 5.9 fold decrease in BCR-ABL and 4.2 fold increase in CCN3 (n = 3, p = 0.001). CCN3 expression was high in normal marrow and blood (range 253–7413 and 120–4280 copies respectively, n = 6). BCR-ABL expression was high in 10 CML patient samples at diagnosis (range 275–319,000 copies). Patients who responded to treatment had low CCN3 expression at diagnosis (range 0–22.1) which increased in response to imatinib (range 53.6–943); patients who had high levels of CCN3 at diagnosis (range 764–3580) did not achieve molecular remission.

CCN3 has a reciprocal relationship with BCR-ABL and provides an additional marker for monitoring clinical response to imatinib.

Mutational activation of p53 suppresses WISP-2/CCN5 signaling in breast tumor cells: a novel circuitry of the progression of cancer

Sushanta Banerjee, Gopal Dhar, Snigdha Banerjee

University of Kansas Medical Center/VA Medical Center

The p53 gene often mutationally activated in human breast cancer. Its activation is associated with the progression of cancer. In contrast, WISP-2/CCN5 has been shown to down regulate in invasive carcinoma cells and tissue samples as compared to the non-invasive conditions where the expression is very high. Therefore, we suggest WISP-2/CCN5 deficiency may be associated with the invasive program. The purpose of this study is to evaluate whether mutant p53 suppresses WISP-2/CCN5 expression in breast tumor cells for the progression of this disease. We found that WISP-2/CCN5 expression is inversely correlated with the mutational activation of p53 in breast tumor cells. Moreover, the studies also found that enforced expression of p53 mutants impaired the expression of WISP-2/CCN5 in non-invasive breast tumor cells. The genetic manipulation of mutant p53 changes the behavior of these cells such as motility. It increased significantly in p53 mediated WISP-2 deficient cells as compared to parent cells that having constitutive expression of WISP-2/CCN5. This change can be imitated by blocking the WISP-2 expression through RNAi or can be reversed by addition of WISP-2 protein in the culture. Collectively, these studies indicate that WISP-2/CCN5 is a negative regulator of breast cancer progression from non-invasive to invasive malignancies.

Role and mechanism of connective tissue growth factor (CCN2/CTGF) induction in osteolytic metastasis of breast cancer

Tsuyoshi Shimo¹, Satoshi Kubota², Norie Yoshioka¹, Soichiro Ibaragi¹, Sachiko Isowa¹, Takanori Eguchi², Akira Sasaki¹, Masaharu Takigawa²

¹Department of Oral and Maxillofacial Surgery, and Biopathological Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and ²Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

Connective tissue growth factor (CCN2/CTGF) mediates angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. Parathyroid hormone-related peptide (PTHrP) is another critical factor in developing osteolytic metastasis. Using both in vivo and in vitro approaches, we investigated whether/how neutralization of CCN2 prevented human breast cancer cell line MDA231-induced bone metastasis, and how PTHrP signaling is related. 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 HUVEC in vitro. Of note, CCN2 expression was specifically observed in PTHrP/ PTH1R-producing cancer cells invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Inhibition of ERK1/2, PKC or PKA by specific inhibitors counteracted the stimulation of ccn2 by PTHrP. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, while TPA, a stimulator of PKC, upregulated it. In conclusion, CCN2 was critically involved in osteolytic metastasis and was induced by PKA and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, N2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

High expression of connective tissue growth factor in paediatric pre-b acute lymphoblastic leukaemia

Joanne M. Boag¹, Alex H. Beesley¹, Martin J. Firth², Joseph R. Freitas¹, Jette Ford¹, David R. Brigstock³, Nicholas H. de Klerk², Ursula R. Kees¹

¹Division of Children’s Leukaemia and Cancer Research, and ²Division of Biostatistics and Genetic Epidemiology, Telethon Institute for Child Health Research, and Centre for Child Health Research, The University of Western Australia, Perth, Western Australia. ³Pediatric Surgery Research Laboratory, Children’s Research Institute, Columbus, Ohio, USA

Gene expression studies have provided new insights into paediatric acute lymphoblastic leukaemia (ALL). Few studies, however, have analysed normal haematopoietic cell populations to identify altered gene expression in ALL. We used oligonucleotide microarrays to compare the gene expression profile of paediatric precursor B (pre-B) ALL specimens with two control cell populations, normal CD34⁺and CD19⁺/IgM⁻cells, in order to focus on genes linked to leukaemogenesis. We identified eight highly deregulated genes, all showing ≥9-fold higher average expression in ALL specimens compared to control cells. These genes were confirmed as significantly deregulated in an independent cohort of 101 ALL specimens. One gene, connective tissue growth factor (CTGF/CCN2), had exceptionally high expression, and this was confirmed in three independent leukaemia studies. Further analysis of CTGF expression in ALL revealed exclusive expression in B-lineage, not T-lineage ALL. Within B-lineage ALL approximately 75% of specimens were consistently positive for CTGF expression, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies by Western blot analysis demonstrated the presence of CTGF in supernatants of ALL cells. These findings indicate that CTGF is secreted by pre-B ALL cells and may play a role in the pathophysiology of this disease.

Role of the ccn family (CCN1, CCN2) in glomerular remodeling and renal disease

Masashi Mukoyama, Hideki Yokoi, Kazutomo Sawai, Kazuwa Nakao

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine

The CCN family proteins are essential for angiogenesis and chondrogenesis, but their role in renal pathophysiology remains unknown. During glomerular remodeling, factors produced by glomerular podocytes may be critically involved. We therefore screened for genes that are expressed in podocytes and are upregulated in anti-Thy-1 glomerulonephritis, a reversible nephropathy model. Using suppression subtractive hybridization, we identified CCN1 among 40 such genes obtained. CCN1 was upregulated specifically at podocytes in early regeneration stages. Recombinant CCN1 enhanced endothelial cell adhesion, but strongly inhibited mesangial cell adhesion and migration. CCN1 also induced podocyte-specific genes, suggesting CCN1 as a podocyte differentiation factor and a mesangial-repellent factor, involved in glomerular remodeling.

CCN2, on the other hand, is a crucial mediator of tubulointerstitial fibrosis, a final common pathway of various nephropathies. We already revealed that CCN2 is a downstream mediator of fibrogenic property of TGF-β. Antisense knockdown of CCN2 in the kidney markedly ameliorated interstitial fibrosis and epithelial-mesenchymal transformation. Furthermore, we generated transgenic mice having podocyte-specific expression of CCN2, which revealed deterioration of glomerular injury. Thus, CCN2 may become a possible target against renal injury progression. Taken together, the CCN family may have a dual modulatory role in renal diseases, i.e., renoprotection and disease progression.

The effects of aminoguanidine on CCN-family member gene expression in the diabetic rat retina

Ingeborg Klaassen¹, John M. Hughes¹, Esther J. Kuiper¹, Paul Canning², Alan W. Stitt², Jan van Bezu³, Casper G. Schalkwijk³, Reinier O. Schlingemann¹

¹Department of Ophthalmology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, ²Department of Ophthalmology, The Queen’s University of Belfast, The Royal Victoria Hospital, Belfast, Northern Ireland, UK., ³Department of Clinical Chemistry and Institute of Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands

The CCN growth factors (Cyr61, CTGF, Nov and WISP-1,-2 and -3) affect cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. Advanced glycation endproducts (AGEs) formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesize that the pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression. Methods: CCN gene expression levels were studied in retinas of control and diabetic rats using real-time quantitative PCR (qPCR) at 6 and 12 weeks of streptozotocin-induced diabetes, with or without aminoguanidine to study the effects of inhibition of endogenous AGE formation. Results: After 6 weeks of diabetes, Cyr61 was upregulated more than 3-fold. At 12 weeks of diabetes, CTGF was upregulated 1.9-fold. Aminoguanidine treatment significantly reduced the Cyr61 and CTGF expression in diabetic rats by 1.5-fold and 1.8-fold, respectively. Immunoblotting for CTGF confirmed these results demonstrating a 1.8-fold increase in protein expression which was abrogated by aminoguanidine treatment. Conclusions: CTGF and Cyr61 are downstream effectors of AGEs in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

Neutralizing antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis model

Poh-Sing NG¹, Koki Endo¹, Norihisa Otake¹, Megumi Iijima¹, Tomohiro Mori¹, Yuka Ikawa², Miki Kondo², Sonoko Chujyo², Kazuhiko Takehara²

¹Nosan Corporation and ²Grad Sch Med Sci, Kanazawa Univ.

Purpose: Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix, and are considered to develop under the influence of certain growth factors. To better understand the mechanisms of persistent fibrosis in SSc, we have investigated whether anti-CTGF neutralizing antibody ameliorates skin fibrosis in our skin fibrosis animal model, which was induced by exogenous TGF beta. Methods: We developed monoclonal antibodies against the native structure of human CTGF using DNA immunization methods. Subsequently, newborn mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified mouse immunoglobulin. The fibrosis observed in our animal model on day 4 was evaluated by the H-E staining. Results: We established monoclonal antibodies recognizing the native conformation of human CTGF. Among them, neutralizing antibody to module 1 and module 2 significantly reduced skin fibrosis in our mice model. Conclusions: Our results indicated that CTGF monoclonal antibody inhibited the fibrosis formation by blocking the TGF beta induced endogenous CTGF of mouse. The result suggests that anti-CTGF neutralizing antibodies are a feasible strategy to treat skin fibrotic diseases. Especially, module 1 and module 2 may be an efficient target of CTGF.

A profibrotic role of CCN2 in the progressive kidney diseases

Hirokazu Okada, Tsutomu Inoue, Hiromichi Suzuki

Department of Nephrology, Saitama Medical School

CCN2 is a down-stream mediator of TGF-β, which is a key growth factor involved in renal fibrogenesis, and as such plays a unique role in mediating mesangial cell/fibroblast proliferation and extracellular matrix production by renal cells. Podocytes, mesangial cells, fibroblasts and endothelial cells produce CCN2 in fibrosing kidneys. We showed that tubular epithelial cells also produced CCN2, which played an essential role in renal fibrogenesis in the murine remnant kidney model and human diabetic nephropathy. Similar to the other cells, an important inducer of CCN2 in epithelial cells is TGF-β. Recently, in addition to BCE-1 and SBE sites in the ccn2 gene promoter that are responsible for TGF-β induction of CCN2 in fibroblasts, we identified a novel, TGF-β-responsive, cis-regulatory element specifically active in the tubular epithelial cells. In my presentation, I will summarize how CCN2 involves in the pathogenesis of progressive kidney diseases and how ccn2 gene expression is regulated in the tubular epithelial cells mainly through data coming up from my laboratory, and mention a possibility of future application for anti-fibrotic therapies.against renal fibrogenesis, a common feature of the progressive kidney diseases.

Overexpression of CCN2 in podocytes deteriorates diabetic nephropathy in diabetic mice

Hideki Yokoi¹, Masashi Mukoyama¹, Kiyoshi Mori¹, Masato Kasahara¹, Kazutomo Sawai¹, Takayoshi Suganami¹, Masao Koshikawa¹, Tetsuro Yoshioka¹, Yoko Saito¹, Akira Sugawara², Kazuwa Nakao¹

¹Kyoto University Graduate School of Medicine, Department of Medicine and Clinical Science and ²National Hospital Organization Kyoto Medical Center, Division of Nephrology

CCN2 is a key mediator of fibrogenic properties of TGF-β. CCN2 mRNA is upregulated in the glomeruli in diabetic nephropathy and CCN2 is thought to be involved in the progression of diabetic nephropathy. To elucidate the role of CCN2 upregulation in podocytes on diabetic nephropathy, we generated transgenic mice (Tg) harboring the mouse CCN2 gene under regulation of human nephrin promoter that is capable of directing podocyte-specific expression. CCN2-Tg had no apparent renal abnormalities at basal conditions. Then, we investigated the progression of renal injury in diabetic CCN2-Tg induced by streptozotocin for 12 weeks. After induction of diabetes, CCN2-Tg exhibited significantly increased urinary albumin excretion by 2.8-fold (p<0.01) compared with WT. Diabetic CCN2-Tg showed increased mesangial expansion and reduced number of podocytes and enhanced vacuolar formation in podocytes by electron microscopic analysis. Immunohistochemical study for podocin revealed disarranged podocin expression in diabetic CCN2-Tg. These results indicate that the overexpression of CCN2 in podocytes can accelerate deterioration of glomerular injuries under diabetic milieu, suggesting that CCN2 plays an important role in the progression of diabetic nephropathy.

Connective tissue growth factor induces myocardial fibrosis, but preserves myocardial function in pressure-induced myocardial hypertrophy

Muhammad Shakil Ahmed¹, Thomas von Luedera¹, Jorgen Gravning¹, Thor Edvardsen¹, Erik Oie², Birthe Mikkelsen¹, Leif Erik Vinge¹, Otto Smiseth², Havard Attramada¹

¹Institute for Surgical Research, Rikshospitalet University Hospital, and Faculty Division Rikshospitalet, University of Oslo, Oslo, Norway and ²Dept. of Cardiology, Rikshospitalet University Hospital, and Faculty Division Rikshospitalet, University of Oslo, Oslo, Norway

Induction of connective tissue growth factor (CTGF) has been demonstrated in heart failure. Yet, the pathophysiologic significance of myocardial CTGF in heart failure remains to be resolved. To elucidate the function of CTGF in the heart, we generated transgenic mice (Tg-CTGF) with cardiac-specific overexpression. Left ventricular end-diastolic pressure (LVEDP) was significantly elevated in Tg-CTGF mice with reduced ventricular compliance. Analysis of myocardial hydroxyproline contents by quantitative HPLC revealed increased myocardial collagen in Tg-CTGF vs. NLC hearts (1.10±0.03 vs.0.82±0.05 pmol/mg; p < 0.05). Consistently, increased myocardial collagen (I) and (III) mRNA levels were found in Tg-CTGF hearts (9.4±0.8 vs. 6.6±0.2 and 8.6±0.5 vs. 5.0±0.1, respectively; p < 0.05). Pressure overload was induced by abdominal aortic banding (AB) in weight-matched male transgenic (CTGF-AB) and non-transgenic mice (NLC-AB). Four weeks after aortic banding, NLC-AB mice displayed overt evidence of cardiac dysfunction. NLC-AB mice displayed significantly increased left ventricular end-diastolic diameter (LVEDD) and LVEDP, and decreased fractional shortening, dP/dtmax, and stroke volume as compared with NLC-sham. Conversely, CTGF-AB mice displayed preserved left ventricular pressure-volume relations. Myocardial CTGF causes interstitial fibrosis with subtle indices of restrictive cardiac dysfunction, but rescues left ventricular function during pressure overload.

The role of CCN2 in the development of cardiac fibrosis and its clinical utility for the diagnosis of heart failure

Masashi Arai¹, Norimichi Koitabashi¹, Kazuo Niwano¹, Atai Watanabe¹, Takashi Nishida², Satoshi Kubota², Masaharu Takigawa², Masahiko Kurabayashi¹

¹Department of Medicine and Biological Science, Gunma University Graduate School of Medicine and ²Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

Purpose: The present study was intended to clarify the role of CCN2 and its functional interactions with brain natriuretic peptide (BNP), an anti-fibrotic peptide, on the development of myocardial fibrosis and diastolic heart failure. Results: In rat pressure-overload cardiac hypertrophy model, relative abundance of mRNA for CCN2 to BNP was correlated with left ventricular stiffness (r = 0.720, P = 0.001), myocardial fibrosis area (r = 0.608, P = 0.002), and procollagen 1 mRNA expression (r = 0.488, P = 0.001). Rats with higher CCN2/BNP ratio had a higher plasma aldosterone concentration. Further investigation demonstrated that CCN2 secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. G-protein coupled receptor ligands induced CCN2 and BNP mRNAs comparably, while aldosterone and TGF-beta preferentially induced expression of CCN2 mRNA. Endomyocardial biopsy from patient with diastolic dysfunction revealed that the abundance of CCN2-positive myocytes was referred to the excessive interstitial fibrosis (N = 46, r = 0.638, P = 0.001) and the acute pulmonary congestion. Plasma CCN2 concentration was significantly elevated in symptomatic patients and correlated with plasma BNP (r = 0.395, P = 0.01). Conclusion: The disproportionate increase in CCN2 relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure. Furthermore, CCN2 may serve as a novel diagnostic modality for heart failure.

Comparison of patterns of expression of CCN5 and CCN2 in rodent tissues

Mark R. Gray¹, Jennifer Malmquist¹, Michael Blea¹, John J. Castellot, Jr^1,2

¹Department of Anatomy and Cell Biology; ²Department of Pharmacology and Experimental Therapeutics; Tufts University School of Medicine, Boston MA, USA

CCN5 is a secreted heparin-and estrogen-regulated matricellular protein that can inhibit vertebrate smooth muscle cell proliferation and motility. CCN5 is expressed throughout murine and human embryonic development in most organs and tissues. This ubiquitous embryonic expression of CCN5 might be a consequence of the hyper-estrogenic intrauterine environment. However, after embryonic development is complete, we expected that CCN5 distribution would be largely restricted to small set of tissues, including smooth muscle cells of the arteries, uterus, airway, and digestive tract. Because CCN5 inhibits proliferation of smooth muscle cells in vitro, it might be necessary to prevent excessive growth in vivo. In contrast, another member of the CCN family, CCN2, promotes smooth muscle cell proliferation in vitro and thus it is expected that its expression levels would be low in uninjured normal adult tissues. Frozen sections from adult tissues and organs were analyzed by immunohistochemistry using anti-CCN5 and anti-CCN2 antibodies. Both CCN5 and CCN2 expression was detected ubiquitously, in the aorta, uterus, bronchioles, and digestive tract as expected, and also in many other adult tissues including the pancreas, spleen, stomach skeletal muscle, ovary, testis, thymus, brain, olfactory epithelium, and kidney. CCN5 and CCN2 expression was found in smooth muscle, endothelial cells, epithelial cells, epithelial cells, skeletal muscle, cells of the nervous system, and numerous other cell types. Rather than having opposite patterns of expression, CCN5 and CCN2 had similar and largely overlapping sites of expression. The wide distribution of both CCN5 and CCN2 suggests that both proteins have additional biological functions beyond those identified in specific cellular and disease models.

Functional analysis of a transgenic mouse expressing reduced levels of CCN5 in an inducible, tissue-specific manner

Michael Blea, John Castellot

Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, Boston MA, USA

Hyperproliferation of smooth muscle cells (SMC) is the hallmark of many important diseases, including arterial restenosis and uterine fibroids. Our examination of agents that could suppress SMC proliferation led to the discovery of CCN5, a heparin-and estrogen-induced gene and protein that strongly inhibits SMC proliferation and motility. To further investigate the role of CCN5 in SMC, a mouse with substantially reduced expression of CCN5 (<25% normal) will be generated by the inducible expression of short hairpin RNA (shRNA) under the control of a tetracycline on/off system. Tissue-specific expression will be achieved using SMC-specific promoters such as SM-22, SMC myosin and a uterine specific promoter. We hypothesize that reduced levels of CCN5 will exacerbate SMC hyperproliferation in mouse models for vascular injury. Supporting this notion is our recent demonstration in mouse, rat and human SMC cell cultures that a short interfering RNA (siRNA) directed against CCN5 strongly inhibits SMC proliferation and motility. Our strategy requires generating two mice, which will then be crossed to produce a functional knockdown of CCN5 that is both inducible and tissue-specific. This approach may also prove useful in generating functional transgenic knockdowns of other proteins in an inducible and tissue-specific manner.

NIH Grant HL49973 and NIH Grant HD046251

Possibility of auricular cartilage reconstruction with CCN2/CTGF

Takuo Fujisawa¹, Kyoji Nakao², Mitsuaki Ono¹, Takako Hattori², Satoshi Kubota², Takuo Kuboki¹, Masaharu Takigawa²

¹Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Department of Oral and Maxillofacial Rehabilitation and ²Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Department of Biochemistry and Molecular Dentistry

Our previous study indicated that CCN2/CTGF might reinforce their own molecular phenotype of chondrocytes derived from different cartilage, such as growth cartilage and articular cartilage. Therefore, in this study, we tried to investigate the effects of CCN2 on auricular chondrocytes proliferation, matrix synthesis, gene expression of differentiated markers and in vivo cartilage formation. Rabbit primary auricular chondrocytes were cultured in the presence or absence of various concentration of recombinant human CCN2. The cell proliferation was evaluated by MTS assay and [³H]-thymidine incorporation, and the proteoglycan synthesis was determined by [³⁵S]-sulfate incorporation. The mRNA levels of chondrocyte differentiation markers were evaluated by real-time RT-PCR, and the accumulation of elastin fibers were visualized by Victoria-blue stain. Cartilage formation was evaluated by in vivo implantation of chondrocytes pellets.

CCN2 stimulated the cell proliferation, DNA synthesis and proteoglycan synthesis. The gene expression of elastin and col 2 were also increased. In addition, the accumulation of elastin fiber was up-regulated. Finally, in vivo implantation experiment indicated that CCN2 strongly accelerated cartilage formation.

This is the first report to show that CCN2 promotes the phenotypes of the auricular chondrocytes, which suggests that CCN2 may be useful for repair or reconstruction of elastic cartilage, e.g., auricular cartilage.

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha 5 beta 1

Takako Hattori¹, Mitsuhiro Hoshijima¹, Eriko Aoyama¹, Miho Inoue¹, Daisuke Araki¹, Hiroshi Hanagata², Akira Miyauchi³, Masaharu Takigawa¹

¹Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and ²R & D Department, Higeta Shoyu Co. Ltd., ³Choshi Laboratory, ProteinExpress Co. Ltd.

CCN2/CTGF is strongly expressed in cartilage, especially in hypertrophic chondrocytes; it modulates chondrocyte differentiation and proliferation and enhances production of extracellular matrices, cell adhesion and migration. To identify CCN2/CTGF interactive proteins which modulate its action, we carried out yeast-two hybrid screening using a cDNA library from the chondrocytic cell line HCS-2/8 and CCN2 as bait. One of the proteins interacting with CCN2/CTGF was fibronectin 1. In the yeast two-hybrid assay we found that only the C-terminal domain (CT) of CCN2/CTGF binds directly to fibronectin 1; the binding enhances adhesion of HCS-2/8 cells to fibronectin 1 in a dose-dependent manner. The same domain has been shown by others to bind to the fibronectin receptors alpha5beta1 and alphavbeta3. A monoclonal antibody against the CT domain partially inhibited CCN2/CTGF adhesion activity, indicating that CCN2/CTGF enhances integrin mediated cell adhesion to fibronectin through its CT domain. CCN2/CTGF enhanced chondrocyte adhesion to fibronectin was abolished by a blocking antibody against alpha5beta1, but not by anti alphavbeta3. These findings suggest that CCN2/CTGF may be trapped by fibronectin 1 in the extracellular matrix and enhances fibronectin binding through the integrin receptor alpha5beta1, but not to alphavbeta3, thereby enhancing alpha5beta1- mediated activation and cell adhesion of chondrocytes.

Effect of CCN2 deletion on the expression of the other ccn family members during chondrocytic terminal differentiation

Harumi Kawaki¹, Satoshi Kubota¹, Akiko Suzuki², Bernard Perbal³, Takeyasu Maeda², Karen M. Lyons⁴, Masaharu Takigawa¹

¹Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutial Sciences and ²Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, ³Laboratoire d’Oncologie Virale et Moléculaire, UFR de Biochimie, Université Paris 7, D. Diderot, ⁴Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, University of California

Among the CCN family members, CCN2 is known as an important regulator in the endochondral ossification, as represented by the disorganized hypertrophic zone in the growth plate of CCN2-mutant mice. In this study, we investigated on the other CCN members during chondrocytic terminal differentiation. Initially, distribution of the other CCN members in developing growth plate cartilage was analyzed. The five CCN members were differentially produced depending upon chondrocytic differentiation stages both in wild-type and the CCN2-mutant mice, although hypertrophic and prehypertrophic zones were enlarged in the CCN2-mutant. Next, we isolated chondrocytes from both mice and induced their differentiation in culture. In the CCN2-mutant chondrocytes, mRNAs of chondrocyte markers showed less increase than in wild-type upon terminal differentiation, and calcification was delayed. Concerning the other five CCN members, CCN2 deletion caused upregulation of CCN3 and downregulation of CCN6 under regular culture conditions. Along with differentiation, the CCN6 mRNA in the CCN2-mutant chondrocytes did not show the increase that was observed in wild-type chondrocytes. In contract, CCN3 expression showed more striking increase in the CCN2-mutant chondrocytes than the wild-type. These results suggest possible role of CCN3 and 6 in the chondrocytic terminal differentiation in collaboration with CCN2.

Expression and possible function of a CCN2 receptor, low density lipoprotein receptor-related protein 1 (LRP1), in chondrocytes

Kazumi Kawata¹, Satoshi Kubota¹, Takanori Eguchi¹, Harumi Kawaki¹, Morihiko Oka², Shogo Minagi², Masaharu Takigawa¹

¹Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and ²Department of Occlusal and Oral Functional Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

LRP1 has been known as an endocytic and signal transmission receptor. LRP1 binds to various ligands, including CCN2, which is a critical factor in endochondral ossification and cartilage regeneration. However, expression and function of LRP1 in normal chondrocytes remains to be investigated. We formerly reported the gene expression and the localization of LRP1 in cartilage and chondrocytes. In the present study, we describe the endocytotic incorporation of CCN2 into chondrocytes and possible roles of LRP1 therein.

Co-localization of endogenous CCN2 and LRP1 was observed on the surface and in the cytoplasm of HCS-2/8. To investigate internalization of CCN2 in chondrocytes, we exogenously added Cy3-labeled recombinant CCN2 (rCCN2) to HCS-2/8 and chased its localization. Increasing internalization of CCN2 was observed along with passing of time, and CCN2 and LRP1 were partially co-localized in HCS-2/8. Interestingly, the internalized rCCN2 was distributed in lysosomes, recycling endosomes and even in the nucleus. To investigate the role of LRP1 in chondrocytes, we treated HCS-2/8 with LRP1 siRNA. Decrease in aggrecan and col2a1 mRNA levels was observed by the siRNA treatment.

These findings suggest that LRP1 plays important roles in the cartilage biology, possibly by regulating the amounts, localities and signals of CCN2 in chondrocytes.

Specific polyclonal antibodies against domains of human CCN3 proteins

Noureddine Lazar, Nathalie Planque and Bernard Perbal

Laboratoire d`Oncologie Virale et Moléculaire, Université Paris 7, D. Diderot, Paris, France

The CCN3 protein inhibits the growth and reduces the tumorigenicity of a large variety of human cancer cell lines, including glioblastoma choriocarcinoma and musculoskeletal Ewing’s sarcoma. The biological importance of truncated isoforms in tumours was established by the identification of an aminotruncated CCN3 protein expressed inMAV-induced nephroblastoma.

The multimodular structure of the CCN3 and other CCN proteins raises interesting questions as to participation of each individual module conferring the biological properties to the full length proteins. Either the biochemical functions of the IGFBP, VWC, TPS, and CT modules contained these proteins are indeed conserved and sum up in the full length protein, or the presence of each module confers on the whole protein specific biological function(s) which may substitute or add upon those of individual modules.

In order to adress the biological functions of each module, either individually or as part of CCN3, we prepared highly specific polyclonal antibodies against each CCN3 modules. Antibody purification was performed by affinity chromatography. The antisera and immunopurified antibodies detect the CCN3 protein expressed in mammalian cells that produce CCN3 naturally or as the result of transfection with expression vector encoding the full length of ccn3 region. Cells transfected with plasmids encoding aminotruncated CCN3 variants were also used in this study. These new polyclonal antibodies should prove useful in analyzing the structure-function relationships within CCN3.

Temporospatial localization of CCN5 protein in murine and human embryos

Jennifer Malmquist, Maria Beatriz Oliveira, John Castellot

Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston MA, USA

CCN5 is highly conserved among vertebrates and is the only CCN family member lacking the C-terminal domain. CCN5 distribution in embryos has not been mapped in a systematic manner. In the adult, CCN5 strongly inhibits smooth muscle cell (SMC) proliferation and motility. The antiproliferative action of CCN5 predicts that it should not be present in an embryonic tissue until the proliferation phase of the morphogenesis of that tissue is complete. To examine this hypothesis, paraffin sections from murine embryos from 9–16 dpc and 4–5mo human embryos were analyzed by immunohistochemistry using affinity-purified anti-peptide CCN5 antibodies. Surprisingly, CCN5 was present in nearly all embryo tissues at the stages analyzed. While CCN5 protein expression is initially present in most tissues, tissue-specific expression increases as embryos age. Expression was particularly strong in vascular tissues, cardiac muscle, myotendonous junctions, intestinal smooth muscle and epithelium, uterus, and bronchiolar epithelium. Expression was absent in bone cartilaginous forms but increasingly expressed as bones undergo ossification. The presence of high levels of CCN5 in proliferating tissues suggests that it functions in a cell-and tissue-specific context, and that the presence of CCN5 is not sufficient by itself to prevent cell proliferation.

NIH Grant HL49973 and NIH Grant HD046251

Promotion of adhesion and migration of human bone marrow stromal cells by CCN2 –mechanism and utility in bone regeneration-

Mitsuaki Ono¹, Satoshi Kubota², Takuo Fujisawa¹, Wataru Sonoyama¹, Harumi Kawaki², Kentaro Akiyama¹, Masamitsu Oshima¹, Takashi Nishida², Masaharu Takigawa², Takuo Kuboki¹

¹Okayama University,Department of Oral and Maxillofacial Rehabilitation and ²Okayama University, Dentistry and Pharmaceutical Sciences

The bone marrow stem cells (BMSCs) are mainly composed of mesenchymal stem cells that are potent in differentiating into different cell types including osteoblasts. Here, we investigated the effects of CCN2 on the adhesion and migration of human BMSC. For cell attachment assay, CCN2 was adsorbed onto plastic or hydroxyapatite (HA) plate prior to cell seeding. Activation of phophorylation signaling pathway upon CCN2-mediated adhesion was also evaluated. Migration of hBMSC was assessed by a modified Boyden chamber method in vitro and with a porous HA/hBMSC hybrid implanted subcutaneously in nude mice in vivo. As a result, CCN2 significantly enhanced the hBMSC attachment, which was neutralized by an anti-integrin αvβ3 antibody. Upon the adhesion mediated by CCN2, p38MAPK was activated and the enhanced cell attachment by CCN2 was neutralized by a p38MAPK inhibitor. This enhancement of cell adhesion was mainly mediated by the C-terminal module of the CCN2. The migration of hBMSC was also stimulated dramatically by CCN2. Moreover, in vivo evaluation indicated that CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HA. These results strongly suggest the utility of CCN2 in the regeneration of an intractable bone defect in combination with HA scaffold and BMSC.

CCN5-induced gene expression in uterine smooth muscle

Joshua Russo, John Castellot

Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston MA, USA

Smooth muscle cells (SMC) in the myometrium of the uterus are involved in a number of uterine pathologies, including leiomyoma (fibroids). Clinical presence of leiomyomas in the general female population is 15–20%, and the prevalence rises to > 60% in black women. To date, the only treatment proven to prevent recurrence of the disease is total abdominal hysterectomy, a procedure performed over 600,0000 times annually in the United States alone. The development of less drastic therapeutic alternatives requires a detailed understanding of the cellular and molecular mechanisms regulating both leiomyoma and normal SMC. Earlier work in our laboratory has shown that primary cultures of neoplastic SMC cells isolated from fibroids display no endogenous production of CCN5 protein, while SMC derived from the myometrium of the same patients display normal levels of CCN5. In addition, forced expression of CCN5 utilizing an adenoviral system inhibits the proliferation and motility of cultured leiomyoma cells. Significantly, neither the upstream nor downstream regulatory elements of CCN5’s biological activities have been examined. The objective of the present study is to use gene microarray technology supplemented by quantitative PCR to determine potential candidates for downstream effector molecules of CCN5 in uterine SMC. Using an adenoviral infection system, CCN5 protein production was upregulated in two patient samples of cultured human uterine myometrial and leiomyoma cells. RNA extracted from the cells was hybridized to a human gene microarray slide containing 21,000 genes. Three separate slides were analyzed for each sample and compared; all displayed highly consistent changes. Preliminary analysis indicates that a total of 505 genes (.024%) display a 3-fold or greater change in the presence of increased CCN5. Prominent among the CCN5-regulated genes are a cluster of approximately 30 extracellular matrix-associated genes. The CCN5-regulated proteins include collagens, MMPs, integrins, and others. To validate the microarray data, quantitative PCR was performed on a number of the regulated genes. While specific interpretation of these results requires further analysis, the pattern of regulated genes is consistent with the anti-proliferative and anti-motility effects caused by CCN5 overexpression and suggests that the ability of CCN5 to regulate a relatively large group of extracellular matrix associated genes represents an important role for this protein class in mediating the mechanism of action of CCN5.

Supported by NIH Grant HD046251

WNT-induced secreted protein 1 (WISP1/CCN4) mRNA splicing variants in normal and transformed chondrocytes

Takeshi Yanagita¹, Satoshi Kubota¹, Kazumi Kawata¹, Harumi Kawaki¹, Teruko Takano-Yamamoto², Shinji Tanaka³, Masaharu Takigawa¹

¹Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan and ²Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, ³Department of Hepato-Biliary-Panccreatic Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan

WISP1/CCN4 is one of the CCN family proteins comprising 6 members. Several reports described a WISP1 variant (WISP1v) lacking the VWC module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue in currently available. In this study, expression and processing of WISP1 mRNA was analyzed in a human chondrosarcoma-derived cell line (HCS-2/8), as well as in other cell lines of different origins. Subsequently, expression of WISP1 mRNA along normal chondrocyte differentiation was similarly analyzed, using rabbit growth cartilage cells (RGC) that were cultured and sampled following a long time course until they were mineralized. Our results showed that WISP1, WISP1v, and a novel variant of WISP1 (WISP1vx) mRNA were expressed in the HCS-2/8 cells. This new variant was lacking the VWC and TSP modules and a part of IGFBP module. This finding suggests a possible role of WISP1vx as well as WISP1v in malignant transformation of chondrocytes. Moreover, we identified WISP1v orthologue as well as that of the full-length mRNA in normal RGC. Interestingly, overexpression of WISP1v in HCS-2/8 cells enhanced the late chondrocytic phenotype of those cells. These results suggest a critical role of WISP1v in chondrocytic differentiation and endochondral ossification.

PROGRAM

October 18, 2006

21:00

Bernard Perbal

Opening remarks: Prospects in the CCN field

Masaharu Takigawa

Welcome and introductory talk

October 19, 2006

Session I: Regulation of Gene Expression

Chairperson: Sandra Irvine

8:30–8:55

Brahim Chaqour

Regulation of the CCN2 gene by mechanical forces in smooth muscle cells

8:55–9:20

Satoshi Kubota

The 3′-untranslated region-mediated regulation of the ccn family genes

9:20–9:45

Herman Yeger

Expression patterns and phenotypic correlations of CCN1-3 in an organotypic model of neurogenesis

Session II: Receptor and signaling pathways

Chairperson: Christian Naus

10:15–10:40

Takashi Nishida

CCN2 (Connective Tissue Growth Factor) supports the binding of fibronectin to alpha5beta1 integrin and maintains integrin signaling.

10:40–11:05

Takako Hattori

Identification of CCN2/CTGF binding proteins from human chondrocytes

11:05–11:30

Ken-ichi Katsube

Notch signal and CCN3 in the bone marrow derived mesenchymal stem cells

Session III: Cell Growth and Differentiation (I)

Chairperson: Ken-ichi Katsube

13:30–13:55

Bernard Perbal

Antiproliferative activity of CCN3: Qualitative and quantitative aspects

13:55–14:20

Wun Chey Sin

Interaction of gap junction protein connexin 43 and CCN3 in breast cancer cells

October 20, 2006

Session IV: Cell Growth and Differentiation (II)

Chairperson: Masaharu Takigawa

9:00–9:25

Christian Naus

Gap junction protein connexin 43 upregulates CCN1 in C6 glioma cells

9:25–9:50

Stephen Twigg

CTGF/CCN2 inhibits adipocyte differentiation

9:50–10:15

Jeniffer Malmquist

CCN5 suppresses restenosis in a mouse vascular injury model

Session V: Development, Remodeling and Regeneration (I)

Chairperson: Bernard Perbal

10:45–11:10

Masaharu Takigawa

Roles of CCN proteins in skeletal growth and regeneration

11:10–11:35

Karen M. Lyons

CCN1 and CCN2 are required for skeletal and vascular development

11:45–12:00

Akira Kudo

Mechanical Stress-related action of NOV (CCN3) together with periostin in the periodontal ligament and periosteum

Session VI: Development, Remodeling and Regeneration (II)

Chairperson: Karen M. Lyons

14:00–14:25

Takuo Kuboki

Alveolar bone regeneration using human bone marrow stromal cells and CCN2:-to attain reliable and sophisticated dental implant therapy

14:25–14:50

Manabu Kanyama

Expression, regulation and function of CCN2 during odontogenesis

14:50–15:15

Teruko Takano-Yamamoto

Expression of CTGF mRNA and induction of apoptosis in osteocytes by mechanical stimulation in mice

15:45–18:00 Poster Session

October 21, 2006

Session VII: Tumor Biology (I)

Chairperson: Luc Desnoyers

8:30–8:55

Ruth Lupu

Involvement OF CCN1: α_Vβ₃ and CCN1: α₆β₁ integrin binding in acquisition of antiestrogen resistance and tumor progression

8:55–9:20

Sandra Irvine

Funcrtional roles of CCN3 in chronic myeloid leukemia

9:20–9:45

Lynn McCallum

CCN3 expression is reduced as a result of Bcr-Abl kinase in chronic myeloid leukaemia

9:45–10:10

Sushanta K. Banerjee

Mutational activation of p53 suppresses WISP-2/CCN5 signaling in breast tumor cells: A novel circuitry of the progression of cancer

Session VIII: Tumor Biology (II)

Chairperson: Ruth Lupu

10:40–11:05

Tsuyoshi Shimo

Role and mechanism of connective tissue growth Factor (CCN2/CTGF) induction in osteolytic metastasis of breast cancer

11:05–11:30

Luc Desnoyers

Hyaluronan and CD44 participate in WISP-1 promoted mammary tumor growth and metastasis

11:30–11:55

Ursula R. Kees

High expression of connective tissue growth factor in paediatric pre-B acute lymphoblastic leukaemia

Session IX: Pathobiology (fibrosis) (I)

Chairperson: Stephen Twigg

13:30–13:55

Masashi Mukoyama

Role of the CCN family (CCN1, CCN2) in glomerular remodeling and renal disease

13:55–14:20

Ingeborg Klaassen

The effects of aminoguanidine on CCN-family member gene expression in the diabetic rat retina

14:20–14:45

Poh-Sing Ng

Neutralizing antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis model

14:45–15:10

Hirokazu Okada

A profibrotic role of CCN2 in the progressive kidney diseases

Session X: Pathobiology (fibrosis) (II)

Chairperson: Herman Yeger

15:40–16:05

Hideki Yokoi

Overexpression of CCN2 in podocytes deteriorates diabetic nephropathy in diabetic mice

16:05–16:30

Muhammad Shakil Ahmed

Connective tissue growth factor induces myocardial fibrosis, but preserves myocardial function in pressure-induced myocardial hypertrophy

16:30–16:55

Masashi Arai

The role of CCN2 in the development of cardiac fibrosis and its clinical utility for the diagnosis of heart failure

16:55–17:10 Concluding Remarks