Getting to the heart of the matter: CCN2 plays a role in cardiomyocyte hypertrophy

Connective tissue growth factor (CTGF/CCN2) is overexpressed in diabetes. Diabetic rats possess myocardial and cardiomyocyte hypertrophy. In a recent report, Wang and colleagues (Am J Physiol Cell Physiol. 2009 Jul 22. [Epub ahead of print]) show that CCN2 directly mediates cardiomyocyte hypertrophy as well as that induced by high glucose and fatty acid. CCN2 acted via the TrkA receptor. These data are the subject of this commentary, and emphasize that CCN2 may be an excellent target for therapy in diabetes.     

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.     

Proteins on the catwalk: modelling the structural domains of the CCN family of proteins

The CCN family of proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) are multifunctional mosaic proteins that play keys roles in crucial areas of physiology such as angiogenesis, skeletal development tumourigenesis, cell proliferation, adhesion and survival. This expansive repertoire of functions comes through a modular structure of 4 discrete domains that act both independently and in concert. How these interactions with ligands and with neighbouring domains lead to the biological effects is still to be explored but the molecular structure of the domains is likely to play an important role in this. In this review we have highlighted some of the key features of the individual domains of CCN family of proteins based on their biological effects using a homology modelling approach.     

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.     

CCN6 (WISP3): a new anti-cancer therapy?

The CCN family of matricellular proteins are dysregulated in cancers, and may strategies targeting them may represent novel approaches to treating these diseases. A recent study from Huang and colleagues (Cancer Res. 70: 3340-50, 2010) suggests that CCN6 (WISP3) is downregulated in aggressive breast cancers, and this phenomenon may result in the promotion of tumor survival. CCN6 may represent a novel therapeutic approach to breast cancer.     

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.     

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.     

Report on the 7th international workshop on the CCN family of genes

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.     

CCN5, a secreted protein,localizes to the nucleus

CCN5, a member of the CCN family of growth factors, inhibits the proliferation and migration of smooth muscle cells in cell culture and animal models. Expressed in both embryonic and adult tissues, CCN5 exhibits a matricellular localization pattern characteristic of secreted proteins that are closely associated with the cell surface. In addition to this observed expression pattern, immunohistochemical evidence suggests the presence of nuclear CCN5 in some cells. To determine if CCN5 localizes to the nucleus we performed immunofluorescence, confocal imaging, and cell fractionation to corroborate the immunohistochemical observations. After confirming the presence of nuclear CCN5 using four independent experimental methods, we identified a single putative nuclear localization signal in the von Willebrand factor C domain of mouse and rat CCN5. Site directed mutagenesis of the three basic amino acids in the putative nuclear localization sequence did not prevent nuclear localization of CCN5 in four different cell types, suggesting that CCN5 nuclear transport is not mediated by the only canonical nuclear localization signal present in the primary amino acid sequence. Future work will address the mechanism of nuclear localization and the function of nuclear versus secreted CCN5.     

Yin and Yang revisited: CCN3 as an anti-fibrotic therapeutic?

Fibrotic diseases are a significant cause of mortality. It is being increasingly appreciated that the cellular microenvironment plays a key role in promoting pathological fibrosis. A previous Bits and Bytes described an elegant series of experiments published by Bruce Riser and colleagues (Am J Pathol. 2009: 174:1725–34) that showed that CCN3 (nov) antagonizes the fibrogenic effects of CCN2.and hence could represent a novel anti-fibrotic therapy. They have continued their excellent work and have recently used the ob/ob mouse as a model of obesity and diabetic nephropathy to show that CCN3 could block the induction of profibrotic gene expression, fibrosis and loss of kidney function (Am J Pathol. 2014;184:2908–21). Also, reversal of fibrosis was observed. Thus this paper provides strong evidence that CCN3 may be used as a novel therapy to treat diabetes caused by obesity.     

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-β1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-β1. Conversely, TGF-β1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-β1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.     

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.     

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.     

Physical interaction of CCN2 with diverse growth factors involved in chondrocyte differentiation during endochondral ossification

CCN family member 2 (CCN2) has been shown to promote the proliferation and differentiation of chondrocytes, osteoblasts, osteoclasts, and vascular endothelial cells. In addition, a number of growth factors and cytokines are known to work in harmony to promote the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification. Earlier we showed that CCN2 physically interacts with some of them, suggesting that multiple effects of CCN2 on various differentiation stages of chondrocytes may be attributed to its interaction with these growth factors and cytokines. However, little is known about the functional interaction occurring between CCN2 and other growth factors and cytokines in promoting chondrocyte proliferation and differentiation. In this study we sought to shed light on the binding affinities between CCN2 and other essential growth factors and cytokines known to be regulators of chondrocyte differentiation. Using the surface plasmon resonance assay, we analyzed the dissociation constant between CCN2 and each of the following: TGF-β1, TGF-β3, IGF-I, IGF-II, PDGF-BB, GDF5, PTHrP, and VEGF. We found a strong association between CCN2 and VEGF, as well as a relatively high association with TGF-β1, TGF-β3, PDGF-BB, and GDF-5. However, the sensorgrams obtained for possible interaction between CCN2 and IGF-I, IGF-II or PTHrP showed no response. This study underlines the correlation between CCN2 and certain other growth factors and cytokines and suggests the possible participation of such interaction in the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification.     

ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts

The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.     

Yin and Yang Part Deux: CCN5 inhibits the pro-fibrotic effects of CCN2

There is no treatment for fibrotic disease is a significant cause of mortality. CCN2 Members of the CCN family of matricellular proteins have a characteristic four domain structure. CCN2 (connective tissue growth factor) is believed to play an essential role in fibrogenesis. In a recent paper, data are provided that CCN5 (wisp2), which lacks the carboxy-terminal heparin-binding domain shared by the other CCN proteins, may act as a dominant-negative protein to suppress CCN2-mediated fibrogenesis. These data are consistent with the notion that different CCN proteins may enhance or suppress each other's action and also suggest that CCN5, may be used as a novel anti-fibrotic therapy.     

CCN2 is not required for skin development

Mice lacking the pro-adhesive matricellular protein connective tissue growth factor (CTGF/CCN2) display an embryonic lethal phenotype due to defects in bone and cartilage. However, the specific role of CCN2 in skin development is unknown. Here, we generated mice deleted for CCN2 in the entire body (using a cre/lox system in which CCN2 is deleted in the entire body due to the presence of a constitutively expressed cre recombinase). We found that CCN2 was not required for the development of skin as defined by skin thickness measurements, trichrome staining and immunostaining with anti-CD31 (to detect endothelial cells) and anti-α−SMA (to detect smooth muscle cells and pericytes) antibodies. Thus, although recently we have shown that CCN2 is required for fibrogenesis in postnatal mice, CCN2 is not required for skin development during embryogenesis.     

Yin and Yang: CCN3 inhibits the pro-fibrotic effects of CCN2

Fibrotic disease is a significant cause of mortality. CCN2 (connective tissue growth factor [CTGF]), a member of the CCN family of matricellular proteins, plays a significant role in driving the fibrogenic effects of cytokines such as transforming growth factor β (TGFβ). It has been proposed that other members of the CCN family can either promote or antagonize the action of CCN2, depending on the context. A recent elegant study published by Bruce Riser and colleagues (Am J Pathol. 174:1725–34, 2009) illustrates that CCN3 (nov) antagonizes the fibrogenic effects of CCN2. This paper, the subject of this commentary, raises the intriguing possibility that CCN3 may be used as a novel anti-fibrotic therapy.