CCN2 Connective Tissue Growth Factor Research Articles

Module 4-Deficient CCN2/Connective Tissue Growth Factor Attenuates the Progression of Renal Fibrosis via Suppression of Focal Adhesion Kinase Phosphorylation in Tubular Epithelial Cells.

CCN2/connective tissue growth factor (CTGF) potentially serves as a therapeutic target for chronic kidney disease. Here we investigated CCN2 module-4, encoded by Ccn2 exon 5, through the generation of Ccn2 exon 5 knockout mice (Ex5-/- mice). To investigate renal fibrosis pathogenesis, Ex5-/- mice were employed to model unilateral ureteral obstruction (UUO), unilateral ischemic-reperfusion injury (UIRI), and 5/6 nephrectomy. Interstitial fibrosis was significantly attenuated in the Ex5-/- mice in the three models. Furthermore, phosphorylated focal adhesion kinase (FAK) levels in tubular epithelial cells were significantly lower in the kidneys of the UUO- and UIRI-Ex5-/- mice than those of the Ex5+/+ mice. Moreover, CCN2 module 4-mediated renal tubule FAK and promoted fibrosis. These findings indicate that CCN2 module-4-FAK pathway components will serve as therapeutic targets for effectively attenuating renal fibrosis.

CCN2/CTGF-A Modulator of the Optic Nerve Head Astrocyte.

In primary open-angle glaucoma (POAG), a neurodegenerative disease of the optic nerve (ON) and leading cause of blindness, the optic nerve head (ONH) undergoes marked structural extracellular matrix (ECM) changes, which contribute to its permanent deformation and to degeneration of ON axons. The remodeling process of the ECM causes changes in the biomechanical properties of the ONH and the peripapillary sclera, which is accompanied by an increased reactivity of the resident astrocytes. The molecular factors involved in the remodeling process belong to the Transforming growth factor (TGF)-β superfamily, especially TGF-β2. In previous publications we showed that TGF-β2 induced ECM alterations are mediated by Cellular Communication Network Factor (CCN)2/Connective Tissue Growth Factor (CTGF) and recently we showed that CCN2/CTGF is expressed by astrocytes of the ON under normal conditions. In this study we wanted to get a better understanding of the function of CCN2/CTGF under normal and pathologic conditions. To this end, we analyzed the glial lamina and peripapillary sclera of CCN2/CTGF overexpressing mice and studied the effect of CCN2/CTGF and increasing substratum stiffness on murine ON astrocytes in vitro. We observed enhanced astrocyte reactivity in the ONH, increased ECM protein synthesis in the peripapillary sclera and increased Ccn2/Ctgf expression in the ONH during the pathologic development in situ. CCN2/CTGF treatment of primary murine ON astrocytes induced a higher migration rate, and increase of ECM proteins including fibronectin, elastin and collagen type III. Furthermore, the astrocytes responded to stiffer substratum with increased glial fibrillary acidic protein, vimentin, actin and CCN2/CTGF synthesis. Finally, we observed the reinforced appearance of CCN2/CTGF in the lamina cribrosa of glaucomatous patients. We conclude that reactive changes in ONH astrocytes, induced by the altered biomechanical characteristics of the region, give rise to a self-amplifying process that includes increased TGF-β2/CCN2/CTGF signaling and leads to the synthesis of ECM molecules and cytoskeleton proteins, a process that in turn augments the stiffness at the ONH. Such a scenario may finally result in a vicious circle in the pathogenesis of POAG. The transgenic CTGF-overexpressing mouse model might be an optimal model to study the chronic pathological POAG changes in the ONH.

Mesenchymal stem cell-derived CCN2 promotes the proliferation, migration and invasion of human tongue squamous cell carcinoma cells.

Recent studies have demonstrated that mesenchymal stem cells (MSC) exhibit a tropism to tumors and form the tumor stroma. In addition, we found that MSC can secrete different types of factors. However, the involvement of MSC‐derived factors in human tongue squamous cell carcinoma (TSCC) growth has not been clearly addressed. The CCN family includes multifunctional signaling molecules that affect the initiation and development events of various tumors. In our study, we report that CCN2/connective tissue growth factor (CTGF) was the most highly induced among the CCN family members in MSC that were co‐cultured with TSCC cells. To evaluate the relationship between CCN2 and TSCC growth, we downregulated MSC‐derived CCN2 expression with shRNA targeting CCN2 and found that MSC‐secreted CCN2 promotes TSCC cell proliferation, migration and invasion. We also confirmed that MSC‐derived CCN2 partially accelerated tumor growth in vitro. Taken together, these results suggest that MSC‐derived CCN2 contributes to the promotion of proliferation, migration and invasion of TSCC cells and may be a possible therapy target in the future.

Role of CCN2 in Amino Acid Metabolism of Chondrocytes.

CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage.

CCN2 as a Novel Molecule Supporting Energy Metabolism of Chondrocytes

CCN2/connective tissue growth factor (CTGF) is a unique molecule that promotes both chondrocytic differentiation and proliferation through its matricellular interaction with a number of extracellular biomolecules. This apparently contradictory functional property of CCN2 suggests its certain role in basic cellular activities such as energy metabolism, which is required for both proliferation and differentiation. Comparative metabolomic analysis of costal chondrocytes isolated from wild-type and Ccn2-null mice revealed overall impaired metabolism in the latter. Among the numerous metabolites analyzed, stable reduction in the intracellular level of ATP, GTP, CTP, or UTP was observed, indicating a profound role of CCN2 in energy metabolism. Particularly, the cellular level of ATP was decreased by more than 50% in the Ccn2-null chondrocytes. The addition of recombinant CCN2 (rCCN2) to cultured Ccn2-null chondrocytes partly redeemed the cellular ATP level attenuated by Ccn2 deletion. Next, in order to investigate the mechanistic background that mediates the reduction in ATP level in these Ccn2-null chondrocytes, we performed transcriptome analysis. As a result, several metabolism-associated genes were found to have been up-regulated or down-regulated in the mutant mice. Up-regulation of a number of ribosomal protein genes was observed upon Ccn2 deletion, whereas a few genes required for aerobic and anaerobic ATP production were down-regulated in the Ccn2-null chondrocytes. Among such genes, reduction in the expression of the enolase 1 gene was of particular note. These findings uncover a novel functional role of CCN2 as a metabolic supporter in the growth-plate chondrocytes, which is required for skeletogenesis in mammals.

Pro-Invasive Activity of the Hippo Pathway Effectors YAP and TAZ in Cutaneous Melanoma

YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway. Both are amplified in many human cancers and promote cell proliferation and epithelial–mesenchymal transition. Little is known about the status of the Hippo pathway in cutaneous melanoma. We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior. YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma. TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential. Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection. YAP knockdown also reduced invasion in a model of skin reconstruct. Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain–dependent transcription and CCN2/CTGF expression. Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.

Novel chondrogenic and chondroprotective effects of the natural compound harmine

A significant number of natural compounds have been shown to regulate the behavior of the cells, in collaboration with cellular proteins. CCN2/connective tissue growth factor (CTGF) has been reported to have essential roles in cartilage development, chondrocyte proliferation and differentiation as well as regulation of the extracellular matrix metabolism. Previous studies demonstrated the capability of CCN2 to regenerate surgical defects in articular cartilage of rat knee. Also, transgenic mice over-expressing cartilage-specific CCN2 were shown to be more resistant to aging-related cartilage degradation. We hypothesized that small molecules that induce CCN2 in chondrocytes could be novel candidates to increase the resistance to aging-related cartilage degradation, or even to correct cartilage degenerative changes incurred in OA. Therefore, this study screened a compound library and identified the β-carboline alkaloid harmine as a novel inducer of CCN2 in human chondrocytic HCS-2/8 cells and osteoarthritic articular chondrocytes. Harmine increased the expression of the cartilage markers aggrecan and COL2α1, as well as that of the master regulator of chondrogenesis, SOX-9. Moreover, harmine notably induced chondrogenesis of prechondrocytic ATDC5 cells in micromass cultures. The chondroprotective effect of harmine was investigated under inflammatory condition by stimulation with TNFα, and harmine was shown to ameliorate TNFα-induced decrease in expression of CCN2 and cartilage markers. These findings uncover novel chondrogenic effects of harmine and indicate harmine as a potential drug for prevention and/or repair of cartilage degradation.

CCN1 and CCN2: blood brothers in angiogenic action

CCN2/connective tissue growth factor (CTGF) is a matricellular protein essential for skeletal development during embryogenesis. In adulthood, aberrant CCN2 expression is associated with many malignancies and fibrosis of virtually every organ. Despite its prominent expression in endothelial cells in the vasculature, the role of CCN2 in vessel development was unknown. In a recent study, Hall-Glenn et al. (PLoS ONE 7:e30562) have revealed the role of CCN2 in developmental angiogenesis through a detailed analysis of how CCN2 mediates the interaction between vascular endothelial cells and pericytes. In addition, CCN2 also regulates endothelial basement membrane formation during vessel formation. Here I compare the angiogenic activities of CCN2 during embryogenesis to those of its homologous family member CCN1 (CYR61), which is essential for cardiovascular development. Understanding the angiogenic actions of CCN1 and CCN2 may have implication in the development of therapeutic strategies targeting these proteins for the treatment of diseases such as cancer and fibrosis.

CCN2/Connective Tissue Growth Factor Is Essential for Pericyte Adhesion and Endothelial Basement Membrane Formation during Angiogenesis

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.

Role of low-density lipoprotein receptor related protein 1 (LRP1) in CCN2/connective tissue growth factor (CTGF) protein transport in chondrocytes

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA

CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3′-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH–RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH–RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis.

Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein.

To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation. Expression of CCN2/CTGF, p-STAT3, p-Smad3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reverse-transcriptase polymerase chain reaction. IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80, and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients' sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3) signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter. CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is down-regulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.

Mechanisms of novel cardioprotective functions of CCN2/CTGF in myocardial ischemia-reperfusion injury

CCN2/connective tissue growth factor (CTGF), a CCN family matricellular protein repressed in healthy hearts after birth, is induced in heart failure of various etiologies. Multiple cellular and biological functions have been assigned to CCN2/CTGF depending on cellular context. However, the functions and mechanisms of action of CCN2/CTGF in the heart as well as its roles in cardiac physiology and pathophysiology remain unknown. Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) were generated and compared with nontransgenic littermate control (NLC) mice. Tg-CTGF mice displayed slightly lower cardiac mass and inconspicuous increase of myocardial collagen compared with NLC mice but no evidence of contractile dysfunction. Analysis of the myocardial transcriptome by DNA microarray revealed activation of several distinct gene programs in Tg-CTGF hearts involved in cardioprotection and growth inhibition. Indeed, Tg-CTGF mice subjected to ischemia-reperfusion injury by in situ transient occlusion of the left anterior descending coronary artery in vivo displayed reduced vulnerability with markedly diminished infarct size. These findings were recapitulated in isolated hearts perfused with recombinant human (h)CTGF before the ischemia-reperfusion procedure. Consistently, Tg-CTGF hearts, as well as isolated adult cardiac myocytes exposed to recombinant hCTGF, displayed enhanced phosphorylation and activity of the Akt/p70S6 kinase/GSK-3β salvage kinase pathway and induction of several genes with reported cardioprotective functions. Inhibition of Akt activities also prevented the cardioprotective phenotype of hearts from Tg-CTGF mice. This report provides novel evidence that CTGF confers cardioprotection by salvage phosphokinase signaling leading to inhibition of GSK-3β activities, activation of phospho-SMAD2, and reprogramming of gene expression.

CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.

CCN family genes in the development and differentiation of cartilage tissues

CCN family is a novel family of proteins consisting of several modulator molecules. The members display a variety of physiological and pathological functions ; hence they are currently attracting the interest of a number of biologists. In terms of the development and regeneration of cartilage tissues, CCN2/connective tissue growth factor (CTGF) is best known among the CCN family members, since it efficiently promotes endochondral ossification and articular cartilage regeneration. Recently, it has been uncovered how CCN2 gene expression is duly regulated along with the differentiation of chondrocytes, which is uncovering the genetic program leading to cartilage tissue development. Moerover, production of other members, such as CCN1 and CCN4, are occasionally observed in chondrocytes, suggesting the contribution of the entire CCN family members to the developmental process of cartilage in vivo.