Bernard Perbal

CCN proteins: multifunctional signalling regulators

CONTEXT Although little is known as yet about the processes that coordinate cell-signalling pathways, matrix proteins are probably major players in this type of global control. The CCN (cyr61, ctgf, nov) proteins are an important family of matricellular regulatory factors involved in internal and external cell signalling. This family participates in angiogenesis, chondrogenesis, and osteogenesis, and they are probably involved in the control of cell proliferation and differentiation. STARTING POINT Runping Gao and David Brigstock (Hepatol Res 2003; 27: 214-20) recently showed that CCN2 (CTGF, connective tissue growth factor) is a cell-adhesion factor for hepatic stellate cells. On exposure to transforming growth factor beta, hepatic stellate cells produce distinct CCN2 isoforms. Gao and Brigstock assign to CCN2 module 3 the capacity to mediate binding to low-density-lipoprotein receptor-related protein (LRP), which was previously reported to interact with CCN2 and to be involved in various types of signalling. They also establish that CCN2 binding to LRP is heparin dependent and that module 4 of CCN2 promotes LRP-independent adhesion of hepatic stellate cells. The differential binding of CCN2 isoforms to LRP highlights the importance of functional interactions between individual modules, and reinforces the concept that different module combinations might confer agonistic or antagonistic activities. WHERE NEXT? It is essential to understand how the distinct configuration of the various CCN isoform affects their biological activities and bioavailability, and to explore the mechanisms and the regulatory processes involved in the production of truncated CCN isoforms. A better understanding of the structural basis for their multifunctionality is a prerequisite to wider use of CCN proteins in molecular medicine.

The CCN family of proteins: structure–function relationships

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure–function relationships.

A structural approach to the role of CCN (CYR61/CTGF/NOV) proteins in tumourigenesis

The CCN (C YR61 [Cystein-rich61]/C TGF [connective tissue growth factor]/N OV [Nephroblastoma overexpressed]) proteins constitute a family of regulatory factors involved in many aspects of cell proliferation and differentiation. An increasing body of evidence indicates that abnormal expression of the CCN proteins is associated to tumourgenesis. The multimodular architecture of the CCN proteins, and the production of truncated isoforms in tumours, raise interesting questions regarding the participation of each individual module to the various biological properties of these proteins. In this article, we review the current data regarding the involvement of CCN proteins in tumourigenesis. We also attempt to provide structural basis for the stimulatory and inhibitory functions of the full length and truncated CCN proteins that are expressed in various tumour tissues.

The Expression of ccn3(nov) Gene in Musculoskeletal Tumors

The CCN3(NOV) protein belongs to the CCN [cysteine-rich CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed gene (Nov)] family of growth regulators, sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Although previous studies have revealed an expression of CCN3 protein in several normal tissues, including kidney, nervous system, lung, muscle, and cartilage, less is known about its expression in tumors. In this study, we analyzed the expression of CCN3 in musculoskeletal tumors, using a panel of human cell lines and tissue samples. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas, in Ewings sarcoma, the expression of this protein seemed to be associated with a higher risk to develop metastases. CCN3 expression was found in 15 of 45 Ewings sarcoma tissue samples. In particular, we did not observe any expression of CCN3 in the 15 primary tumors that did not develop metastases. In contrast, 15 of the 30 primary tumors that developed lung and/or bone metachronous metastases showed a high expression of the protein (P < 0.001, Fishers test). Our studies indicate that CCN3 is generally expressed in the cells of the musculoskeletal system. This protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. Thus, as reported for other CCN genes, the biological properties and regulation of expression of CCN3 are dependent on the cellular context and the nature of the cells in which it is produced. Further studies will help to clarify the biological role of this protein in musculoskeletal neoplasms.

Cooperative Regulation of Chondrocyte Differentiation by CCN2 and CCN3 Shown by a Comprehensive Analysis of the CCN Family Proteins in Cartilage

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

In Ewing's sarcoma CCN3(NOV) inhibits proliferation while promoting migration and invasion of the same cell type

Altered expression of CCN3 has been observed in a variety of musculoskeletal tumours, including Ewings sarcoma (ES). Despite its widespread distribution, very little is known about its biological functions and molecular mechanisms of action. We transfected CCN3 gene into a CCN3-negative ES cell line and analysed the in vitro and in vivo behaviours of stably transfected clones. Forced expression of CCN3 significantly reduced cell proliferation in vitro, growth in anchorage-independent conditions, and tumorigenicity in nude mice. Despite the antiproliferative effect, CCN3-transfected ES cells displayed increased migration and invasion of Matrigel. The decreased expression of α2β1 integrin receptor and the increased amount of cell surface-associated matrix metalloproteinase (MMP)-9 following the expression of CCN3 may be the basis for the increased migratory abilities of transfected cells. Cells lacking α2β1 are less facilitated to have stable anchorage since the predominant collagen extracted from ES tissue is indeed type I collagen, and proMMP-9 was recently found to provide a cellular switch between stationary and migratory ES cell phase. Our findings are in line with those recently obtained in glioblastoma. However, the underlying molecular mechanisms appear to be different, further highlighting the importance of the cellular context in the regulation of function of CCN proteins.

CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

CCN3 Inhibits Neointimal Hyperplasia Through Modulation of Smooth Muscle Cell Growth and Migration

Objective—CCN3 belongs to the CCN family, which constitutes multifunctional secreted proteins that act as matrix cellular regulators. We investigated the pathophysiological roles of CCN3 in the vessels. Methods and Results—We examined the effects of CCN3 on the proliferation and migration of rat vascular smooth muscle cells (VSMC). CCN3 knockout mice were created, and vascular phenotypes and neointimal hyperplasia induced by photochemically induced thrombosis were investigated. CCN3 suppressed the VSMC proliferation induced by fetal bovine serum. The neutralizing antibody for transforming growth factor-&bgr; did not affect the growth inhibitory effect of CCN3. Moreover, CCN3 enhanced the mRNA expression of cyclin-dependent kinase inhibitors, p21 and p15. Gamma secretase inhibitor, an inhibitor of Notch signaling, partially inhibited the enhanced expression of p21 induced by CCN3. CCN3 also inhibited the VSMC migration. Finally, the histopathologic evaluation of the arteries 21 days after the endothelial injury revealed a 6-fold enhancement of neointimal thickening in the null mice compared with the wild-type mice. Conclusion—CCN3 suppresses neointimal thickening through the inhibition of VSMC migration and proliferation. Our findings indicate the involvement of CCN3 in vascular homeostasis, especially on injury, and the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.

The CCN family of genes: a perspective on CCN biology and therapeutic potential

The CCN family of genes currently comprises six secreted proteins (designated CCN1–6 after Cyr61/CCN1; ctgf/CCN2; Nov/CCN3; WISP1/CCN4; WISP2/CCN5, WISP3/CCN6) with a similar mosaic primary structure. It is now well accepted that CCN proteins are not growth factors but matricellular proteins that modify signaling of other molecules, in particular those associated with the extracellular matrix. CCN proteins are involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Since their first identification as matricellular factors, the CCN proteins now figure prominently in a variety of major diseases and are now considered valid candidates for therapeutic targeting. Dissection of the molecular mechanisms governing the biological properties of these proteins is being actively pursued by an expanding network of scientists around the globe who will meet this year at the 5th International Workshop on the CCN family of Genes, organized by the International CCN Society (http://ccnsociety.com), home for an international cadre of collaborators working in the CCN field.