Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio-marker of fibroproliferative disease?
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ABSTRACT:
SUBMITTER: Leask A
PROVIDER: S-EPMC2721078 | biostudies-other | 2009 Jun
REPOSITORIES: biostudies-other
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Project description:Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.
| S-EPMC8377001 | biostudies-literature
Project description:Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGF? luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.
| S-EPMC3561339 | biostudies-literature
Project description:Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.
| S-EPMC2747032 | biostudies-literature
Project description:Fibrotic disorders are the end point of many chronic diseases in different tissues, where an accumulation of the extracellular matrix occurs, mainly because of the action of the connective tissue growth factor (CTGF/CCN2). Little is known about how this growth factor activity is regulated. We found that decorin null myoblasts are more sensitive to CTGF than wild type myoblasts, as evaluated by the accumulation of fibronectin or collagen III. Decorin added exogenously negatively regulated CTGF pro-fibrotic activity and the induction of actin stress fibers. Using co-immunoprecipitation and in vitro interaction assays, decorin and CTGF were shown to interact in a saturable manner with a K(d) of 4.4 nM. This interaction requires the core protein of decorin. Experiments using the deletion mutant decorin indicated that the leucine-rich repeats (LRR) 10-12 are important for the interaction with CTGF and the negative regulation of the cytokine activity, moreover, a peptide derived from the LRR12 was able to inhibit CTGF-decorin complex formation and CTGF activity. Finally, we showed that CTGF specifically induced the synthesis of decorin, suggesting a mechanism of autoregulation. These results suggest that decorin interacts with CTGF and regulates its biological activity.
| S-EPMC3129205 | biostudies-literature
Project description:Reduced production of type I procollagen is a prominent feature of chronologically aged human skin. Connective tissue growth factor (CTGF/CCN2), a downstream target of the transforming growth factor-beta (TGF-beta)/Smad pathway, is highly expressed in numerous fibrotic disorders, in which it is believed to stimulate excessive collagen production. CTGF is constitutively expressed in normal human dermis in vivo, suggesting that CTGF is a physiological regulator of collagen expression. We report here that the TGF-beta/Smad/CTGF axis is significantly reduced in dermal fibroblasts, the major collagen-producing cells, in aged (> or = 80 years) human skin in vivo. In primary human skin fibroblasts, neutralization of endogenous TGF-beta or knockdown of CTGF substantially reduced the expression of type I procollagen mRNA, protein, and promoter activity. In contrast, overexpression of CTGF stimulated type I procollagen expression, and increased promoter activity. Inhibition of TGF-beta receptor kinase, knockdown of Smad4, or overexpression of inhibitory Smad7 abolished CTGF stimulation of type I procollagen expression. However, CTGF did not stimulate Smad3 phosphorylation or Smad3-dependent transcriptional activity. These data indicate that in human skin fibroblasts, type I procollagen expression is dependent on endogenous production of both TGF-beta and CTGF, which act through interdependent yet distinct mechanisms. Downregulation of the TGF-beta/Smad/CTGF axis likely mediates reduced type I procollagen expression in aged human skin in vivo.
| S-EPMC2877594 | biostudies-literature
Project description:Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.
| S-EPMC4524627 | biostudies-literature
Project description:CCN2/connective tissue growth factor is highly expressed in hypertrophic chondrocytes and is required for chondrogenesis. However, the transcriptional mechanisms controlling its expression in cartilage are largely unknown. The activity of the Ccn2 promoter was, therefore, investigated in osteochondro-progenitor cells and hypertrophic chondrocytes to ascertain these mechanisms. Sox9 and T-cell factor (TCF) x lymphoid enhancer factor (LEF) factors contain HMG domains and bind to related consensus sites. TCF x LEF factors are normally repressive but when bound to DNA in a complex with beta-catenin become activators of gene expression. In silico analysis of the Ccn2 proximal promoter identified multiple consensus TCF x LEF elements, one of which was also a consensus binding site for Sox9. Using luciferase reporter constructs, the TCF x LEF x Sox9 site was found to be involved in stage-specific expression of Ccn2. Luciferase, electrophoretic mobility shift assay (EMSA), and ChIP analysis revealed that Sox9 represses Ccn2 expression by binding to the consensus TCF x LEF x Sox9 site. On the other hand, the same assays showed that in hypertrophic chondrocytes, TCF x LEF x beta-catenin complexes occupy the consensus TCF x LEF x Sox9 site and activate Ccn2 expression. Furthermore, transgenic mice in which lacZ expression is driven under the control of the proximal Ccn2 promoter revealed that the proximal Ccn2 promoter responded to Wnt signaling in cartilage. Hence, we propose that differential occupancy of the TCF x LEF x Sox9 site by Sox9 versus beta-catenin restricts high levels of Ccn2 expression to hypertrophic chondrocytes.
| S-EPMC2934638 | biostudies-literature
Project description:Pre-osteoblast adhesion and interaction with extracellular matrix (ECM) proteins through integrin receptors result in activation of signaling pathways regulating osteoblast differentiation. Connective tissue growth factor (CTGF/CCN2) is a matricellular protein secreted into the ECM. Prior studies in various cell types have shown that cell adhesion to CTGF via integrin receptors results in activation of specific signaling pathways that regulate cell functions, such as differentiation and cytoskeletal reorganization. To date, there are no studies that have examined whether CTGF can serve as an adhesive substrate for osteoblasts. In this study, we used the MC3T3-E1 cell line to demonstrate that CTGF serves as an adhesive matrix for osteoblasts. Anti-integrin blocking experiments and co-immunoprecipitation assays demonstrated that the integrin αvβ1 plays a key role in osteoblast adhesion to a CTGF matrix. Immunofluorescence staining of osteoblasts cultured on a CTGF matrix confirmed actin cytoskeletal reorganization, enhanced spreading, formation of focal adhesions, and activation of Rac1. Alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining demonstrated that osteoblast attachment to CTGF matrix enhanced maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves integrin-mediated activation of specific signaling pathways, we performed Western blot, chromatin immunoprecipitation (ChIP) and qPCR assays. Osteoblasts cultured on a CTGF matrix showed increased total and phosphorylated (activated) forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Inhibition of ERK blocked osteogenic differentiation in cells cultured on a CTGF matrix. There was an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter, and in the expression of osteogenic markers regulated by Runx2. Collectively, the results of this study are the first to demonstrate CTGF serves as a suitable matrix protein, enhancing osteoblast adhesion (via αvβ1 integrin) and promoting cell spreading via cytoskeletal reorganization and Rac1 activation. Furthermore, integrin-mediated activation of ERK signaling resulted in increased osteoblast differentiation accompanied by an increase in Runx2 binding to the osteocalcin promoter and in the expression of osteogenic markers.
| S-EPMC4340870 | biostudies-literature
Project description:BackgroundThe regulatory mechanisms of the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2) in human articular chondrocytes have not been clarified. We investigated the effect of prostaglandin E2 (PGE2) on CTGF/CCN2 expression in chondrocytes.FindingsArticular cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were isolated and cultured in vitro. Chondrocytes were stimulated with PGE2, PGE receptor (EP)-specific agonists, or interleukin (IL)-1. CTGF expression was analyzed using quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The inhibitory effects of EP receptor antagonists (for EP2 and EP4) against PGE2 stimulation were also investigated. Stimulation of chondrocytes with PGE2 or IL-1 significantly suppressed CTGF expression. The suppressive effect of PGE2 was reproduced by EP2/EP4 receptor agonists but not by EP1/EP3 receptor agonists, and was partially blocked by an EP4 receptor antagonist, suggesting that the EP4 receptor has a dominant role.ConclusionsPGE2 may be involved in the regulation of CTGF/CCN2 expression in human articular chondrocytes via the EP4 receptor. Elucidation of EP4-mediated signaling in chondrocytes may contribute to a better understanding of the effects of PGE2 in arthritis.
| S-EPMC2826353 | biostudies-literature
Project description: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.
| S-EPMC3610707 | biostudies-literature