The CCN2/CTGF interactome: an approach to understanding the versatility of CCN2/CTGF molecular activities

Cellular communication network 2 (CCN2), also known as connective tissue growth factor (CTGF) regulates diverse cellular processes, some at odds with others, including adhesion, proliferation, apoptosis, and extracellular matrix (ECM) protein synthesis. Although a cause-and-effect relationship between CCN2/CTGF expression and local fibrotic reactions has initially been established, CCN2/CTGF manifests cell-, tissue-, and context-specific functions and differentially affects developmental and pathological processes ranging from progenitor cell fate decisions and angiogenesis to inflammation and tumorigenesis. CCN2/CTGF multimodular structure, binding to and activation or inhibition of multiple cell surface receptors, growth factors and ECM proteins, and susceptibility for proteolytic cleavage highlight the complexity to CCN2/CTGF biochemical attributes. CCN2/CTGF expression and dosage in the local environment affects a defined community of its interacting partners, and this results in sequestration of growth factors, interference with or potentiation of ligand-receptor binding, cellular internalization of CCN2/CTGF, inhibition or activation of proteases, and generation of CCN2/CTGF degradome products that add molecular diversity and expand the repertoire of functional modules in the cells and their microenvironment. Through these interactions, different intracellular signals and cellular responses are elicited culminating into physiological or pathological reactions. Thus, the CCN2/CTGF interactome is a defining factor of its tissue- and context-specific effects. Mapping of new CCN2/CTGF binding partners might shed light on yet unknown roles of CCN2/CTGF and provide a solid basis for tissue-specific targeting this molecule or its interacting partners in a therapeutic context.     

Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins

Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces. These forces are significantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic overload. Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems. Two immediate early gene-encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine-rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2), are highly expressed in several mechanical stress-related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton. Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events. In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibrotic diseases. At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated by various components of the signaling machinery, i.e., small Rho GTPases, mitogen-activated protein kinases, and actin binding proteins. This review discusses the mechanical regulation of the Cyr61 and CTGF in various tissues and cell culture models with a special attention to the cytoskeletally based mechanisms involved in such regulation.     

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.     

Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient’s skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.     

Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates α1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.     

Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes

Connective tissue growth factor (CTGF), also known as CCN2, is implicated in fibrosis through both extracellular matrix (ECM) induction and inhibition of ECM degradation. The role of CTGF in inflammation in cardiomyocytes is unknown. In some mesenchymal cell systems, CTGF mediates effects through TGF-β or tyrosine kinase cell surface receptor, TrkA, signalling. In this study, cellular mechanisms by which CTGF regulates pathways involved in fibrosis and inflammation were explored. Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced. CTGF treatment also increased pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-β1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-β neutralizing antibody and Alk 5 inhibitor (SB431542). A specific blocker of TrkA activation, k252a, also abrogated CTGF-induced effects on fibrosis and gene expresison of MCP-1 and IL-8, but not TNF-α or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-β pathway signalling and partly through TrkA.     

The potential role of ORM2 in the development of colorectal cancer

Colorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Therefore, there is a compelling need for the identification of blood biomarkers that can enable early detection of CRC. We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of 10 patients with CRC compared to healthy volunteers. Enzyme-Linked Immunosorbnent Assay (ELISA) and Western blot were used for further validation. In our quantitative proteomics analysis, we detected 75 human plasma proteins with more than 95% confidence using iTRAQ labeling in conjunction with microQ-TOF MS. 9 up-regulated and 4 down-regulated proteins were observed in the CRC group. The ORM2 level in plasma was confirmed to be significantly elevated in patients suffering from CRC compared with the controls. ORM2 expression in CRC tissues was significantly increased compared with that in corresponding adjacent normal mucous tissues (P<0.001). ITRAQ together with Q-TOF/MS is a sensitive and reproducible technique of quantitative proteomics. Alteration in expression of ORM2 suggests that ORM2 could be used as a potential biomarker in the diagnosis of CRC.     

A single bout of exercise with high mechanical loading induces the expression of Cyr61/CCN1 and CTGF/CCN2 in human skeletal muscle.

High mechanical loading was hypothesized to induce the expression of angiogenic and/or lymphangiogenic extracellular matrix (ECM) proteins in skeletal muscle. Eight men performed a strenuous exercise protocol, which consisted of 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion. Muscle biopsies were taken 30 min and 48 h postexercise from the vastus lateralis muscle and analyzed for the following parameters: mRNA and protein expression of ECM-associated CCN proteins [cysteine-rich angiogenic protein 61 (Cyr61)/CCN1, connective tissue growth factor (CTGF)/CCN2], and mRNA expression of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1alpha. The mRNA expression of Cyr61 and CTGF increased 30 min after the exercise (14- and 2.5-fold, respectively; P < 0.001). Cyr61 remained elevated 48 h postexercise (threefold; P < 0.05). The mRNA levels of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or hypoxia-inducible factor-1alpha did not change significantly at either 30 min or 48 h postexercise; however, the variation between subjects increased markedly in VEGF-A and VEGF-B mRNA. Cyr61 protein levels were higher at both 30 min and 48 h after the exercise compared with the control (P < 0.05). Cyr61 and CTGF proteins were localized to muscle fibers and the surrounding ECM by immunohistochemistry. Fast fibers stained more intensively than slow fibers. In conclusion, mechanical loading induces rapid expression of CCN proteins in human skeletal muscle. This may be one of the early mechanisms involved in skeletal muscle remodeling after exercise, since Cyr61 and CTGF regulate the expression of genes involved in angiogenesis and ECM remodeling.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.     

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Electronic supplementary material

The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users.     

Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth.     

CCN5/WISP-2: A micromanager of breast cancer progression

The gain of plasticity by a subset of cancer cells is a unique but common sequence of cancer progression from epithelial phenotype to mesenchymal phenotype (EMT) that is followed by migration, invasion and metastasis to a distant organ, and drug resistance. Despite multiple studies, it is still unclear how cancer cells regulate plasticity. Recent studies from our laboratory and others’ proposed that CCN5/WISP-2, which is found intracellularly (in the nucleus and cytoplasm) and extracellularly, plays a negative regulator of plasticity. It prevents the EMT process in breast cancer cells as well as pancreatic cancer cells. Multiple genetic insults, including the gain of p53 mutations that accumulate over the time, may perturb CCN5 expression in non-invasive breast cancer cells, which ultimately helps cells to gain invasive phenotypes. Moreover, emerging evidence indicates that several oncogenic lesions such as miR-10b upregulation and activation of TGF-β-signaling can accumulate during CCN5 crisis in breast cancer cells. Collectively, these studies indicate that loss of CCN5 activity may promote breast cancer progression; application of CCN5 protein may represent a novel therapeutic intervention in breast cancer and possibly pancreatic cancer.     

Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.