CCN3: Doctor Jekyll and Mister Hyde

CCN proteins are key regulators of signaling pathways that are essential for the control of normal life, from birth to death. As such, they make use of their unique mosaic structure to interact with several other regulatory proteins and ligands that control the fate of living cells. The various functions attributed to the CCN proteins may sometimes appear contradictory, but this situation reflects the complexity of the multimolecular scaffolds in which CCN proteins are engaged and the critical impact of the microenvironment that dictates the bioavailability of the elementary building blocks. CCN3 is one of the best examples of a CCN protein showing biological properties which may at first glance appear opposite or contradictory. Indeed, CCN3 acts both as a tumor suppressor and is associated with higher metastatic potential. Furthermore, the physical interaction of CCN3 with VEGF and its potential antiangionenic activity in glioma cells are in apparent contradiction with its proangiogenic activity in rabbit cornea. In this communication, I am revisiting the observations that led us to these apparent contradictions. After pointing out how the methodologies that were employed might have contributed to the confusion, I briefly discuss the dual biological activities of CCN3 in the context of tumor cell engineering and survival prognosis.     

CCN proteins: A centralized communication network

The CCN family of proteins includes six members presently known as CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6. These proteins were originally designated CYR61, CTGF, NOV, and WISP-1, WISP-2, WISP-3. Although these proteins share a significant amount of structural features and a partial identity with other large families of regulatory proteins, they exhibit different biological functions. A critical examination of the progress made over the past two decades, since the first CCN proteins were discovered brings me to the conclusion that most of our present knowledge regarding the functions of these proteins was predicted very early after their discovery. In an effort to point out some of the gaps that prevent us to reach a comprehensive view of the functional interactions between CCN proteins, it is necessary to reconsider carefully data that was already published and put aside, either because the scientific community was not ready to accept them, or because they were not fitting with the « consensus » when they were published. This review article points to avenues that were not attracting the attention that they deserved. However, it is quite obvious that the six members of this unique family of tetra-modular proteins must act in concert, either simultaneously or sequentially, on the same sites or at different times in the life of living organisms. A better understanding of the spatio-temporal regulation of CCN proteins expression requires considering the family as such, not as a set of single proteins related only by their name. As proposed in this review, there is enough convincing pieces of evidence, at the present time, in favor of these proteins playing a role in the coordination of multiple signaling pathways, and constituting a Centralized Communication Network. Deciphering the hierarchy of regulatory circuits involved in this complex system is an important challenge for the near future. In this article, I would like to briefly review the concept of a CCN family of proteins and critically examine the progress made over the past 10 years in the understanding of their biological functions and involvement in both normal and pathological processes.     

CCN5: biology and pathophysiology

CCN5 is one of six proteins in the CCN family. This family of proteins has been shown to play important roles in many processes, including proliferation, migration, adhesion, extracellular matrix regulation, angiogenesis, tumorigenesis, fibrosis, and implantation. In this review, we focus on the biological and putative pathophysiological roles of CCN5. This intriguing protein is structurally unique among the CCN family members, and has a unique biological activity profile as well.     

7th international workshop on the CCN family of genes: Nice to be in nice

In this Editorial, I would like to provide our readers with a brief mid-year update about our activities and efforts to bring together researchers working on intercellular signaling proteins at international meetings. The roots emerged about 20 years ago in the discovery of three genes originally designated cyr61, ctgf, and nov. The proteins encoded by these genes were first proposed to constitute a family of proteins (CCN) which now comprises 6 members (CCN1, CCN2, CCN3, CCN4-6) including the wisp proteins. These proteins were recognized to share a striking structural organization and a high degree of identity although they exhibited quite distinct biological properties. After historical considerations regarding the reasons for using the CCN acronym, and how the ICCNS publishing landscape that drove the ICCNS from Cell Communication and Signaling to the Journal of Cell Communication and Signaling, this short update will focus on the 7th edition of the International Workshop on the CCN family of genes to be held in Nice, Oct 16–19, 2013.     

Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein

The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.     

CCN3 and calcium signaling

The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.     

Association of the metastatic phenotype with CCN family members among breast and oral cancer cells

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

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

The CCN (CYR61 [Cystein-rich61]/CTGF [connective tissue growth factor]/NOV [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.     

Alternative splicing of CCN mRNAs …. it has been upon us

Variant CCN proteins have been identified over the past decade in several normal and pathological situations. The production of CCN truncated proteins have been reported in the case of CCN2(ctgf), CCN3(nov), CCN4(wisp-1) and CCN6(wisp-3). Furthermore, the natural CCN5 is known to miss the C-terminal domain that is present in all other members of the CCN family of proteins. In spite of compelling evidence that assign important biological activities to these truncated CCN variants, their potential regulatory functions have only recently begun to be widely accepted. The report of CCN1(cyr61) intron 3 retention in breast cancer cells now confirms that, in addition to well documented post-translational processing of full length CCN proteins, alternative splicing is to be regarded as another effective way to generate CCN variants. These observations add to a previous bulk of evidence that support the existence of alternative splicing for other CCN genes. It has become clearly evident that we need to recognize these mechanisms as a means to increase the biological diversity of CCN proteins.     

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.     

Role of CCN,a vertebrate specific gene family,in development

The CCN family of genes constitutes six members of small secreted cysteine rich proteins, which exists only in vertebrates. The major members of CCN are CCN1 (Cyr61), CCN2 (CTGF), and CCN3 (Nov). CCN4, CCN5, and CCN6 were formerly reported to be in the Wisp family, but they are now integrated into CCN due to the resemblance of their four principal modules: insulin like growth factor binding protein, von Willebrand factor type C, thrombospondin type 1, and carboxy‐terminal domain. CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues, but most studies have focused on their principal role in osteo/chondrogenesis and vasculo/angiogenesis from the aspect of migration, growth, and differentiation of mesenchymal cells. CCN proteins simultaneously integrate and modulate the signals of integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch by direct binding. However, the priority in the use of the signals is different depending on the cell status. Even the equivalent counterparts show a difference in signal usage among species. It may be that the evolution of the CCN family continues to keep pace with vertebrate evolution itself.     

The CCN family: a new class of inflammation modulators?

Uncontrolled or sustained inflammation is the underlying cause of or actively contributes to the progression of many chronic pathologies such as atherosclerosis, arthritis, or neuroinflammatory diseases. Matricellular proteins of the CCN family (CYR61/CTGF/NOV) have emerged as localized multitasking signal integrators. These structurally conserved secreted proteins specifically interact with and signal through various extracellular partners, in particular integrins, which enable them to play crucial roles in various processes including development, angiogenesis, wound healing and diseases such as fibrosis, vascular disease and cancer. In this review, we discuss the possibility that the CCN family members could represent a putative new class of modulators of inflammation. In this context, we focused on their relationship with cytokines and chemokines. In vitro, CCN expression is finely regulated by diverse inflammatory mediators including cytokines (TNFα, IL1β, TGF-β), small factors such as prostaglandins, nitric oxide, histamine and serotonin, and extracellular matrix enzymes. In addition, CCN proteins acting alone or in concert with their specific partners appear to be potent regulators of the production of cytokines and chemokines in a context-dependent manner. Finally, emerging studies suggest a potential role for CCN proteins in chronic inflammatory diseases such as atherosclerosis, rheumatoid arthritis, inflammatory kidney diseases and neuroinflammatory pathologies such as Alzheimer’s disease. CCN members could therefore represent new potential therapeutic targets for drug development against such diseases.     

Anti-fibrotic effect of CCN3 accompanied by altered gene expression profile of the CCN family

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

CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC

Nephroblastoma overexpressed gene encodes a matricellular protein (CCN3/NOV) of the CCN family, comprising CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins are involved in the regulation of mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration in multiple cell types. Compared to CCN2/CTGF, known as a profibrotic protein, the biological role of CCN3/NOV in liver fibrosis remains obscure. In this study we showed ccn3/nov mRNA to increase dramatically following hepatic stellate cell activation, reaching peak levels in fully transdifferentiated myofibroblasts. In models of experimental hepatic fibrosis, CCN3/NOV increased significantly at the mRNA and protein levels. CCN3/NOV was found mainly in non-parenchymal cells along the areas of tissue damage and repair. In the bile-duct ligation model, CCN3/NOV was localized mainly along portal tracts, while the repeated application of carbon tetrachloride resulted in CCN3/NOV expression mainly in the centrilobular areas. In contrast to CCN2/CTGF, the profibrotic cytokines platelet-derived growth factor-B and -D as well as transforming growth factor-β suppressed CCN3/NOV expression. In vitro, CCN3/NOV siRNA attenuated migration in the cirrhotic fat storing cell line CFSC well in line with in vivo findings that various types of cells expressing CCN3/NOV migrate into the area of tissue damage and regeneration. The suppression of CCN3/NOV enhanced expression of profibrotic marker proteins, such as α-smooth muscle actin, collagen type I, fibronectin, CCN2/CTGF and TIMP-1 in primary rat hepatic stellate cells and in CFSC. We further found that adenoviral overexpression of CCN2/CTGF suppressed CCN3/NOV expression, while the overexpression of CCN3/NOV as well as the suppression of CCN3/NOV by targeting siRNAs both resulted in enhanced CCN2/CTGF expression. These results indicate the complexity of CCN actions that are far beyond the classic Yin/Yang interplay.     

CCN3 modulates bone turnover and is a novel regulator of skeletal metastasis

The CCN family of proteins is composed of six secreted proteins (CCN1-6), which are grouped together based on their structural similarity. These matricellular proteins are involved in a large spectrum of biological processes, ranging from development to disease. In this review, we focus on CCN3, a founding member of this family, and its role in regulating cells within the bone microenvironment. CCN3 impairs normal osteoblast differentiation through multiple mechanisms, which include the neutralization of pro-osteoblastogenic stimuli such as BMP and Wnt family signals or the activation of pathways that suppress osteoblastogenesis, such as Notch. In contrast, CCN3 is known to promote chondrocyte differentiation. Given these functions, it is not surprising that CCN3 has been implicated in the progression of primary bone cancers such as osteosarcoma, Ewing’s sarcoma and chondrosarcoma. More recently, emerging evidence suggests that CCN3 may also influence the ability of metastatic cancers to colonize and grow in bone.