Report on the First International Workshop on the CCN Family of Genes

J Clin Pathol: Mol Pathol 2001;54:105–107 105 Report on the ﬁrst international workshop on the CCN family of genes Mol Path: first published as 10.1136/mp.54.2.105 on 1 April 2001. Downloaded from

The first international workshop on the CCN family of ingrowth into the growth plate, it probably plays an impor- genes brought together 75 participants coming from tant role in many aspects of bone growth and repair. essentially all laboratories working in this expanding field. Expression of ctgf in newt limb regeneration with or The meeting was a great success, with exceptionally good without denervation was described in a poster presented by quality scientific exchange, and many fruitful personal Moussad et al. Early ctgf expression was detected in diVer- contacts being made. ent cell types. A role for CTGF was proposed in morpho- As can be seen in this meeting summary, major progress genesis (digit indentation of stump) and hypertrophy of has been made in the fields of development and pathobiol- cartilage. Increased ctgf expression after denervation ogy. New aspects concerning the structural basis for some resulted in muscle atrophy. In this system, CTGF was functions of the CCN proteins and their conservation shown to play a role in the inflammatory response, emerged and most certainly constitute exciting avenues for extracellular matrix remodelling, cell proliferation, endo- future research in the CCN field. chondral ossification, and morphogenetic cell death. CTGF-L/rCOP-1/WISP-2 also appears to be implicated Role of the CCN proteins in developmental in osteoblast and chondrocyte function because it is found processes Several reports have confirmed the importance of CCN in high amounts in osteoblasts and stimulates adhesion and proteins in the chondrogenesis and ossification processes. mineralisation of osteoblasts and proteoglycan synthesis in Early studies by L Lau and collaborators had established chondrocytes (presented by S Kumar). that CYR61 was involved in chondrogenesis. The results NOV also appears to be involved in chondrocyte that were presented at this meeting indicated that cyr61 is function and diVerentiation because it is found in the expressed in trophoblasts and spongioblasts and estab- growth plate as well as in endochondromas, where expres- lished a role for CYR61 in the formation of the chorion and sion was generally higher in low rather than high grade its vascularisation, as shown by the eVects of the lesions. In chondrocyte organ cultures, nov expression was recombinant CYR61 protein on cell proliferation, migra- stimulated by parathyroid hormone related protein tion, and attraction of endothelial cells (as shown in in vitro (PTHrP), a regulator of growth plate chondroctye cultures or grafts). Cyr61 knockout of mice resulted in diVerentiation (presented by B Alman). In addition, nov is embryonic lethality as a result of vascular defects in both expressed during the first trimester in the developing embryonic and extraembryonic tissues. musculoskeletal system including the spinal cord, skeletal A major role for CYR61 appears to be in bone signalling. and smooth muscle, and chondrocytes (presented by P It is produced by mesenchymal cells and osteoclasts from Schofield). fracture callus, in matured growth plate chondrocytes, in At early stages of development in the chick embryo, the http://mp.bmj.com/ proliferating osteoblast cell lines, and in primary cultures. expression of nov was detected in dorsal and periventricu- Moreover, expression of cyr61 is stimulated in osteoblasts lar aspects of the neural tube, from 2 days onwards by vitamin D3 and growth factors, all of which are impor- (presented by K Katsube). With this exception, nov tant in bone metabolism (presented by N Scuhtze). expression was restricted to mesodermally derived struc- Results presented by M Takigawa established that tures (predominant in somites and myotomes in areas from connective tissue growth factor (CTGF; highly expressed which the hypaxial musculature originates). NOV was also in chondrocytes, and designated Hcs28 by the authors) is found in the notochord, in the apical ridges of limb buds, on September 27, 2021 by guest. Protected copyright. involved in skeletogenesis not only during prenatal and in the posterior part of branchial arches, particularly in development but also under traumatic conditions and in the second arch. Cos7 cells expressing nov and grafted into bone fracture repair. The eVects of recombinant CTGF the flank of the embryo induced a supernumerary limb and corresponding antibodies on proliferation and diVer- bud-like structure. In ovo electroporation experiments entiation of chondrocytes (proteoglycans and collagen showed that nov is regulated by Wnt1 (reminiscent of synthesis), osteoblasts (mineralisation), and endothelial WISP genes being targets of Wnt regulation). This cells (proliferation, migration, and angiogenesis) were supports an involvement of NOV in ectomesenchymal assessed using in vitro cultures. A paracrine eVect of interactions with a possible dorsalising eVect. CTGF on three cell lines indicated involvement in As in the chick, the expression of nov is essentially endochondral ossification. The role of CTGF in fracture repair suggests future clinical applications. detected in musculoconnective tissues in the lateral part of In a similar manner to CYR61, CTGF also figures dermomyotomes in early mouse embryos and later on in prominently in vascularisation (endothelial cells and myo- the hypaxial muscles, ocular muscles, and blood vessel cardium) and cartilage development (presented by K walls (presented by E Andermacher). The pax3 and nov Lyons). CTGF is strongly expressed in skeletal growth expression patterns suggested that PAX3 and NOV are plates, in particular, in the context of chondrocyte functionally connected in hypaxial myogenesis. Overex- hypertrophy, a necessary maturation step for endochondral pression of nov in embryonic stem (ES) cells in vitro ossification. Overexpression of ctgf after transfection inter- stimulates cell proliferation and survival and early fered with the proliferation of stromal cells in the bone myogenesis, but blocks terminal muscular diVerentiation marrow, articular chondrogenesis, and cartilage hypertro- and the formation of myotubes. Similarly, chimaeric mouse phy, depending on the degree of expression and in associ- fetuses, obtained from the fusion of normal and nov+ ES ation with transforming growth factor â (TGF-â). Further cells, presented with severe malformation in the craniobra- confirmation was obtained from ctgf knockout studies in chial and limb muscles. The systems involved in the nega- mice, where lack of CTGF led to skeletal defects aVecting tive regulation of nov at late stages of myogenesis that allow ribs, sternum, cervical vertebrae, and craniofacial ele- the terminal diVerentiation of muscle are currently being ments. Because CTGF might also induce vascular studied.

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The eVects of NOV on in vitro muscle diVerentiation function during pathological conditions such as inflamma- were exemplified by the capacity of full length mouse NOV tion, wound healing, and atherosclerosis. protein to induce a block of diVerentiation of 10T1/2 The expression of the WISP genes had been reported to Mol Path: first published as 10.1136/mp.54.2.105 on 1 April 2001. Downloaded from fibroblasts, as opposed to truncated NOV, which induced be regulated by Wnt. In addition to nov being another tar- complete diVerentiation (poster presented by D Tahri). get of Wnt regulation (see above), a novel gene, wise, bear- DiVerential expression of NOV was also observed in satel- ing a weak homology to the CCN family members may lite stem cells undergoing muscle diVerentiation, and in play a role in the Wnt signalling pathway because muscle regeneration (poster presented by Cherel et al). immunoprecipitation studies suggest it binds to the Wnt A striking example for the conservation of CCN protein receptor Frizzled (presented by N Itasaki ). W Kutz exam- functions throughout species was provided by studies per- ined the expression pattern of WISP-3 in eukaryotic cells. formed with Xenopus laevis (presented by B Latinkic). Previous work has shown that loss of function mutations in Cloning and sequencing of cyr61 in this species had WISP-3 are found in patients with the heritable skeletal revealed a high phylogenetic conservation of this gene. In disorder progressive pseudorheumatoid dysplasia, impli- situ hybridisation allowed the detection of maternal cyr61 cating a role for WISP-3 in skeletal growth and homeosta- RNAs up to the cleavage stage and embryonic ones from sis. the neurulation stage, essentially in the dorsal aspect of the A large number of reports now indicate that ctgf expres- embryo in mesodermal structures first (somites, noto- sion is regulated by TGF-â in many instances. However, an chord, branchial arch mesenchyme). Later on (from the tail increasing body of evidence suggests that this relation is not bud stage) the expression was also detected in ectodermal obligatory. A TGF-â response element that is distinct from tissues (otic vesicles and neural tube) and finally in the the previously identified TGF-â response element was dis- heart of the tadpole. A miss-expression of the cyr61 gene cussed. A Leask showed that the induction of CTGF by (complete or partial) induced by injections in the ventral TGF-â is repressed by tumour necrosis factor á (TNF-á) regions of the embryo caused several malformations, through a nuclear factor êB (NFêB) dependent pathway, pointing to a role for CYR61 in the dorsalisation processes and that the regulation of ctgf by TGF- involves SMAD of the early embryo, possibly interfering with the wnt path- â binding to another distinct TGF- responsive element. way. In addition, the CYR61 domains were diVerentially â implicated: domains 1 and 2 in the axial malformations and CTGF expression in punch biopsies was strongly corre- development of ectopic muscle and domain 4 in the blast- lated with the presence of granulation tissue fibroblasts, opore closure. peaking around seven to 10 days after injury. During the S Morais da Silva presented the characterisation of a new granulation phase, CTGF expression was correlated CCN-like gene and its role in an original system of “post- temporally and spatially with TGF-â expression by natal organogenesis”; the regeneration of a sectioned limb fibroblasts, rather than TGF-â expression by inflammatory in the newt. This CCN-like gene, 70/71, was expressed in cells during the first few days after injury (presented by N the blastema several days after the lesion and retinoic acid Hayashi). Gingival overgrowth in patients receiving pheny- treatment, with a heterogenous proximodistal distribution, toin, but not nifedipine or cyclosporin, showed high CTGF supporting the hypothesis for the role of this gene in the expression, suggesting that diVerent molecular mecha- proximodistal identity of the blastema cells and possibly in nisms underlie the fibrotic reaction to each drug. In http://mp.bmj.com/ epithelial–mesenchymal interactions. cultured gingival cells, TGF-â increases collagen mRNA, Although more distantly related, both tsg (twisted lysyl oxidase mRNA values and activity, and CTGF gastrulation) and sog (short gastrulation) drosophila production. However, CTGF itself only increased the proteins share sequence homology with members of the amount of insoluble collagen and lysyl oxidase activity, CCN family. L Marsh reported that expression of the tsg without concomitant increases in their mRNAs, showing gene can enhance the antagonistic activity of sog towards that CTGF complements the activities of TGF-â but does not mediate all the profibrogenic eVects of TGF- bone morphogenetic proteins (BMPs). Interestingly, tsg â on September 27, 2021 by guest. Protected copyright. homologues have been cloned from zebrafish and mamma- (presented by P Trackman). lian species, and have been shown to be functionally equiv- A similar relation between CTGF and TGF-â occurs in alent. mesangial cells (MCs): CTGF appears to stimulate MC migration and TGF-â induced fibronectin production, but not á smooth muscle actin (áSMA) production or cell pro- CCN proteins and signalling liferation, which are induced by TGF-â and platelet New results were also oVered concerning the potential derived growth factor (PDGF), respectively (presented by receptor and signalling pathways involving CCN proteins. I Bloom). Induction of CTGF in MCs and human renal T Grzeszkiewicz presented data describing the integrin receptors that are involved in CYR61 induced activities in fibroblasts is stimulated in a protracted manner by TGF-â and transiently by lysophatidyic acid, the latter involving primary human skin fibroblasts. Integrin á6â1 and heparan sulphate proteoglycans were found to mediate the heptahelical receptors. CTGF expression by both stimuli adhesion of fibroblasts to CYR61. CYR61 induced migra- was dependent on members of the Rho family as well as an tion of fibroblasts was mediated by integrin ávâ5, whereas intact cytoskeleton (presented by M Goppelt-Struebe). integrin ávâ3 is required for the enhancement of basic Rho, together with protein kinase C and phosphatidylinosi- fibroblast growth factor (bFGF) stimulated fibroblast tol 3-kinase (PI-3 kinase), also appears to be involved in mitogenesis. These findings suggest that CYR61 induces mediating CYR61 production in smooth muscle cells diVerent biological eVects through its interaction with dis- (SMCs) that have been exposed to stretch forces. Whereas tinct integrin receptors. ctgf and cyr61 are serum inducible immediate early genes in SMCs, the application of stress forces to cultured SMCs In a similar study, presented by S Lam, integrin áMâ2 was found to be necessary for the adhesion of peripheral blood results in a rapid and pronounced but transient increase in monocytes and THP-1 cells to CYR61 or CTGF. The cyr61 expression, but protracted downregulation of ctgf observation that both CTGF and CYR61 proteins are gene expression. Thus cyr61 and ctgf are diVerentially found in advanced atherosclerotic lesions and wounds regulated by mechanical forces and might have distinct suggests that the interaction of monocytes with these pro- roles in processes such as mechanotransduction or pheno- teins might have important implications for monocyte typic modulation (presented by B Chaqour).

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Structural basis for the biological activities of the TGF-â2, and TGF-â3 was observed. The role of CTGF in CCN proteins renal ﬁbrosis was also reported by B Riser. Mesangial cells

Structural studies concerning CCN proteins are blossom- can be stimulated by TGF-â, a high glucose concentration, Mol Path: first published as 10.1136/mp.54.2.105 on 1 April 2001. Downloaded from ing. As stated in the introductory session by B Perbal, the or cyclic stretch to express CTGF which, in turn, can multimodular structure of the CCN protein presents a increase the production of fibronectin and collagen type I. remarkable biochemical challenge and identifying CCN A 27-fold increase in CTGF expression was also observed protein partners should permit the fundamental questions in db/db mice after 3.5 months of diabetes as compared regarding the biological activities of these proteins to be with the normal db/m control animals. In a similar vein, the answered. urinary CTGF concentrations were measured in patients G Grotendorst presented data showing that the DNA with diabetic nephropathy, and it was suggested that synthesis promoting activity of CTGF in NRK fibroblasts CTGF values may be a useful predictor of patients is absolutely dependent upon the presence of another destined for progressive nephropathy and endstage disease. growth factor, epidermal growth factor (EGF). This find- V Paradis described a rat model of CCl4 induced liver ing is consistent with similar observations made with fibrosis, where CTGF expression was correlated with the CYR61, thus showing that these CCN proteins are not, by late stages of septal fibrosis and cirrhosis. Furthermore, in themselves, mitogenic growth factors. CTGF can be a group of 39 liver biopsies from patients with chronic cleaved by plasmin into N-terminal and C-terminal hepatitis C, 21 cases showed moderate or strong CTGF domains. Whereas the N-terminal domain was found to expression, as did 21 of 28 liver biopsies from patients with exert eVects on collagen synthesis, the C-terminal domain chronic liver diseases, irrespective of aetiology. modulates DNA synthesis. The potential role of CTGF in systemic sclerosis, or Proteolytically processed CTGF fragments of 16– scleroderma, was discussed by K Takehara. CTGF 20 kDa containing the C-terminal domain (module IV) concentrations were found to increase in the sera of have been observed in porcine uterine flushings by D Brig- patients with scleroderma compared with normal controls. stock. These fragments can be produced in a cell culture Subcutaneous injections of TGF-â into mice resulted in system using Chinese hamster ovary (CHO) cells trans- the transient formation of granulation tissue that disap- fected with an expression vector that encodes the full pears over several days. Injection of TGF-â and CTGF, length protein. however, resulted in persistent fibrotic tissue formation. A L Desnoyers presented data showing that the newly two step fibrosis model was proposed in which TGF-â identified CCN family member, WISP-1, interacts directly induces fibrosis, whereas CTGF is involved in the mainte- with decorin and biglycan. WISP-1 was found to possess a nance of fibrosis. specific binding site for dermatan sulphate that is responsi- ble for its interaction with these dermatan sulphate proteoglycans. These data suggest that decorin and Conclusions biglycan may modulate WISP-1 activity in tumours where Taken together, the results presented at the first inter- these proteins are overexpressed. national workshop on CCN proteins have highlighted some exciting aspects of the functions of CCN genes.

Pathobiology of CCN proteins If not primary players, the CCN proteins probably have http://mp.bmj.com/ R Lupu reported that CYR61 expression was detected in a role as eVectors during the embryonic and fetal develop- about 30% of invasive human breast tumour biopsies. ment of nerves, muscle, bone marrow, and bone. Expression of cyr61 is greatly increased in heregulin trans- The widespread expression of the CCN genes in a diver- fected MCF7 breast cancer cells, and anti-CYR61 sity of cell types also suggests a complexity of molecular antibodies abolish the invasiveness and migration of these functions yet to be discovered. That CCN proteins act in a cells in vitro. Moreover, CYR61 is suYcient to confer the large variety of tissue and cell types and across a broad oestrogen independence and anti-oestrogen resistance of species range should generate increased interest in their breast cancer cells. These results suggest that CYR61 study. on September 27, 2021 by guest. Protected copyright. might play an important role in breast cancer progression. C Wenger observed that both cyr61 and ctgf mRNAs are The first international workshop on the CNN family of genes was supported by moderately overexpressed in chronic pancreatitis and grants from Ligue Nationale Contre le Cancer (Comité du Cher), Université highly overexpressed in pancreatic cancer. Paris - D. Diderot, Munin Corp, Fibrogen, Genentech, Glaxo Wellcome, and The pattern of nov expression was established in a series Promega. of diVerent tumours. H Yeger described refined methods of C AYER-LELIEVRE analysing NOV expression in normal and tumoral human Faculté de Medecine, 87025 Limoges, France tissues by means of in situ hybridisation and immunocyto- chemistry. It was found that a brief treatment of tissue sec- D BRIGSTOCK tions in the microwave resulted in enhanced signals for the Department of Surgery, Ohio State University, Colombus, Ohio 43205, detection of NOV in both cases. In some cases, the degree USA of NOV expression was found to correlate with increased L LAU tumorigenicity and proliferative capacity (as presented by Department of Molecular Genetics, University of Illinois, Chicago, Manara, and reported in posters by Cadot et al, Glukhova IL 60607, USA et al), whereas in other cases, such as Wilms’s tumours and D PENNICA neuroblastomas, the expression of NOV correlated with Department of Molecular Oncology, Genetech, San Francisco, CA 94080 tumour diVerentiation (as presented by B Perbal and by H USA Yeger). The expression of NOV in adrenocortical adeno- B PERBAL mas and carcinomas was variable, without a clearcut Laboratoire d’Oncologie Virale et Moleculaire, association between levels of expression and tumour stages UFR de Biochimie,Université Paris 7-D. Diderot, (reported in a poster by Kyurkchiev et al). 2 Place Jussieu, 75005 Paris, France Y Ito reported that CTGF expression is strongly [email protected] upregulated in human proliferative and fibrogenic renal H YEGER disease. In culture, induction of CTGF in rat mesangial Department of Paediatric Laboratory Medicine, cells and glomerular visceral epithelial cells by TGF-â1, Hospital for Sick Children, Toronto, Ontario M5G 1XG, Canada