Report on the 7(th) international workshop on the CCN family of genes : October 16-19, 2013-Nice, France.

In this report, chairs of the 7th International Workshop on the CCN family of Genes, review the progress made in understanding the biological functions of CCN proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) with a particular focus on their implications in various pathological conditions, including cancer, fibrosis, diabetes, and cardiovascular diseases.

Report on the fifth international workshop on the CCN family of genes.

The Fifth International Workshop on the CCN Family of Genes was held in Toronto October 18-22, 2008. This bi-annual workshop provides a unique opportunity for the presentation and discussion of cutting edge research in the CCN field. The CCN family members have emerged as extracellular matrix associated proteins which play a crucial role in cardiovascular and skeletal development, fibrosis and cancer. Significant progress has been made in the development of model systems to tease apart the CCN signalling pathways in these systems. Results presented at the conference suggest that targeting these pathways now shows real promise as a therapeutic strategy.

Downregulation of CCN3 expression as a potential mechanism for melanoma progression.

Coculture of human melanocytes with keratinocytes upregulates CCN3, a matricellular protein critical to maintenance of normal homeostasis of melanocytes in the skin. CCN3 affects two fundamental features of melanocyte physiology: it inhibits melanocyte proliferation and stimulates their adhesion to the basement membrane. Here we report that expression of CCN3 is downregulated in advanced melanomas. Aggressive melanoma cell lines did not respond to treatment with CCN3 inducers, such as interleukin-1beta (IL-1beta), while less aggressive melanoma cell lines responded similarly to melanocytes. Immunostaining analyses revealed that CCN3 was present in melanoma cells close to the epidermal-dermal interface, but not in melanoma cells that had invaded deep into the dermis or had metastasized to lymph nodes. Contrary to our expectations, overexpression of CCN3 in 1205Lu metastatic melanoma cells did not affect their adhesion to collagen IV. However, CCN3 decreased the transcription and activation of matrix metalloproteinases and suppressed the invasion of 1205Lu melanoma cells. These results suggest that the lack of CCN3 in advanced melanoma cells contributes to their invasive phenotype. Whereas major matricellular proteins, such as osteopontin, tenascin or secreted protein acidic and rich in cysteine (SPARC), are strongly upregulated in melanoma cells; CCN3 is the first member of this family that is downregulated.

CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.

CCN proteins affect cell proliferation, migration, attachment, and differentiation. We identified CCN3 as a suppressed gene following platelet-derived growth factor (PDGF)-BB or -DD stimulation in a cDNA-array analysis of mesangial cells. In vitro growth-arrested mesangial cells overexpressed and secreted CCN3, whereas the addition of the recombinant protein inhibited cell growth. Induction of mesangial cell proliferation by PDGF-BB or the specific PDGF beta-receptor ligand PDGF-DD led to downregulation of CCN3 mRNA, confirming the array study. Specific PDGF alpha-receptor ligands had no effect. CCN3 protein was found in arterial smooth muscle cells, the medullary interstitium, and occasional podocytes in the healthy rat kidney. Glomerular CCN3 was low prior to mesangial proliferation but increased as glomerular cell proliferation subsided during mesangioproliferative glomerulonephritis (GN). Inhibition of PDGF-B in mesangioproliferative disease led to overexpression of glomerular CCN3 mRNA. CCN3 localized mostly to podocytes in human glomeruli, but this expression varied widely in different human glomerulonephritides. Glomerular cell proliferation negatively correlated with CCN3 expression in necrotizing GN. Our study identifies CCN3 as an endogenous inhibitor of mesangial cell growth and a modulator of PDGF-induced mitogenesis.

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation.

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.

CCN3-mutant mice are distinct from CCN3-null mice.

A recent manuscript reported phenotypic alterations associated to the expression of a CCN3 protein deleted for the Von Willebrand type C repeat that is common to the various members of the CCN family of proteins. In this comment, the biological significance of these alterations is briefly discussed.

Report on the second international workshop on the CCN family of genes.

For the second time, researchers from leading laboratories in the CCN field gathered in Saint-Malo, France, to participate in the Second International Workshop on the CCN family of genes. In addition to the regular research communications, meeting highlights included the inauguration of the first CCN newsletter (http://ccnnewsletter.free.fr) and the recognition of the International CCN Society (http://www.ccnsociety.jussieu.fr) as an important medium for the exchange of scientific knowledge and resources in the CCN field. Once more, the high quality of scientific communications and individual interactions set the stage for an extremely fruitful meeting.

Proposal for a unified CCN nomenclature.

A proposal is put forth to unify the nomenclature of the CCN family of secreted, cysteine rich regulatory proteins. In the order of their description in the literature, CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) constitute a family of matricellular proteins that regulate cell adhesion, migration, proliferation, survival, and differentiation, at least in part through integrin mediated mechanisms. This proposal is endorsed by the International CCN Society and will serve to eliminate confusion from the multiple names that have been given to these molecules.

Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells.

AIMS: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation. METHODS: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences. RESULTS: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity. CONCLUSIONS: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.

Alterations of the MDV oncogenic regions in an MDV transformed lymphoblastoid cell line.

AIMS: Lymphoblastoid cell lines derived from Marek's disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells. METHODS: Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens. RESULTS: In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek's disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences. CONCLUSIONS: Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.

A role for CCN3 (NOV) in calcium signalling.

AIMS: In animals and humans increased expression of CCN3 (NOV) is detected in tissues where calcium is a key regulator, such as the adrenal gland, central nervous system, bone and cartilage, heart muscle, and kidney. Because the multimodular structure of the CCN proteins strongly suggests that these cell growth regulators are metalloproteins, this study investigated the possible role of CCN3 in ion flux and transport during development, control of cell proliferation, differentiation, and pathobiology. METHODS: The isolation of CCN3 partners was performed by means of the two hybrid system. Yeasts were cotransfected with an HL60 cDNA library fused to the transactivation domain of the GAL4 transcription factor, and with a plasmid expressing CCN3 fused to the DNA binding domain of GAL4. Screening of the recombinant clones selected on the basis of leucine, histidine, and tryptophan prototrophy was performed with a beta-galactosidase assay. After the interaction between CCN3 and its putative partners was checked with a GST (glutathione S-transferase) pull down assay, the positive clones were identified by cloning. To establish whether the CCN3 protein affected calcium ion flux, a dynamic imaging microscopy system was used, which allowed the fluorometric measurement of the intracellular calcium concentration. The proteins used in the assays were GST fused with either CCN3 or CCN2 (CTGF) and GST alone as a control. RESULTS: The two hybrid system identified the S100A4 (mts1) calcium binding protein as a partner of CCN3 and the use of the GST fusion proteins showed that the addition of CCN3 and CCN2 to G59 glioblastoma and SK-N-SH neuroblastoma cells caused a pronounced but transient increase of intracellular calcium, originating from both the entry of extracellular calcium and the mobilisation of intracellular stores. CONCLUSIONS: The interaction of CCN3 with S100A4 may account, in part, for the association of CCN3 with carcinogenesis and its pattern of expression in normal conditions. The increased intracellular calcium concentrations induced by CCN3 and CCN2 both involve different processes, among which voltage independent calcium channels might be of considerable importance in regulating the calcium flux associated with cell growth control, motility, and spreading. These observations assign for the first time a biological function to the CCN3 protein and point out a broader role for the CCN proteins in calcium ion signalling.

Patterns of specific genomic alterations associated with poor prognosis in high-grade renal cell carcinomas.

A series of 13 sporadic renal cell carcinomas was analyzed for the specific chromosome rearrangements after serial xenografting into immunodeficient mice. Seven tumors displayed genetic traits of the conventional subtype and 5 showed genetic features of the papillary subtype. In all the xenografted conventional tumors, we observed loss of 3p, as well as loss of the 9p21 region and of the long arm of chromosome 14, both considered as markers of a poor prognosis. In the xenografted papillary tumors, a duplication of chromosome arm 8q was observed concomitant with the duplication of the 7q31 region. The association of the 7q31 and 8q22 approximately qter duplicated regions was also observed for one conventional tumor. The latency of tumor take was found to be reduced and the median time to passage statistically shorter for all tumors which presented the associated duplication of the 7q31 and 8q22 approximately qter regions. The proto-oncogene NOV (nephroblastoma overexpressed gene) maps to 8q24.1 and is overexpressed in some Wilms tumors. It could be an interesting candidate gene, since its level of expression and release in the culture medium was found to be increased in all of the fast growing tumors analyzed.

Inhibition of glioma cell growth and tumorigenic potential by CCN3 (NOV).

AIMS: To establish whether the ectopic expression of CCN3 (NOV) in glioma cells can interfere with their tumorigenic potential and assess its potential value in molecular medicine. METHODS: Glioma cell lines were used to assess whether differences in the degree of intracellular communication induced by the expression of the gap junction protein connexin 43 (Cx43) is related to the differential expression of CCN3 (NOV). The antiproliferative activity of rat CCN3 (rCCN3; NOV) in glioma cells, has been assessed both in vitro and in vivo with glioma cell lines expressing different amounts of CCN3 (NOV). RESULTS: Upon ectopic expression of Cx43, the growth of C6 glioma cells is decreased. An increase of CCN3 (NOV) expression matches the reduced tumorigenic potential of these transfected cells. The localisation of CCN3 (NOV) is affected by the increased expression of Cx43 in the Cx-13 transfected cells, in which it is detected at areas of cell-cell contact. In a xenograft model, CCN3 (NOV) transfected glioma cells were found to induce tumours to a lesser degree than their parental counterparts, which do not express detectable amounts of CCN3 (NOV). CONCLUSIONS: Previous observations had suggested an inverse relation between CCN3 (NOV) expression in glioma cells and their tumorigenicity. These results establish a direct association between the establishment of functional gap junctional intercellular communication and the expression of rCCN3 (NOV). In addition to a negative effect on murine and human cell growth, CCN3 (NOV) has antiproliferative activity on tumour cells in vivo. Thus, the antiproliferative activity of the CCN3 (NOV) protein might involve reorganisation of cellular contacts that play a crucial role in tumorigenesis. The antiproliferative activity of CCN3 (NOV) established in this work sets the stage for the potential use of CCN proteins in molecular oncology.

Expression of the human NOV gene in first trimester fetal tissues.

NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.