CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes

OBJECTIVES: CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo. METHODS: Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [(3)H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis. RESULTS: CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type II and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF. CONCLUSIONS: These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction of elastic cartilage.     

Expression and localization of connective tissue growth factor (CTGF/Hcs24/CCN2) in osteoarthritic cartilage

OBJECTIVE: The investigation of the expression and localization of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) in normal and osteoarthritic (OA) cartilage, and quantification of CTGF/Hcs24-positive cells. METHODS: Cartilage samples of patients (n=20) with late stage OA were obtained at total joint replacement surgery. Morphologically normal cartilage was harvested for comparison purposes from the femoral heads of 6 other patients with femoral neck fracture. Paraffin-embedded sections were stained by Safranin O. The severity of the OA lesions was divided into four stages (normal, early, moderate, and severe). The localization of protein and mRNA for CTGF/Hcs24 was investigated by immunohistochemistry and in situ hybridization, respectively. The population of CTGF/Hcs24-positive chondrocytes in OA cartilage and chondro-osteophyte was quantified by counting the number of the cells under light microscopy. RESULTS: Signals for CTGF/Hcs24 were detected in a small percentage of chondrocytes throughout the layers of normal cartilage. In early stage OA cartilage, the CTGF/Hcs24-positive chondrocytes were localized mainly in the superficial layer. In moderate to severe OA cartilage, intense staining for CTGF/Hcs24 was observed in proliferating chondrocytes forming cell clusters next to the cartilage surface. In chondro-osteophyte, strong signals were found in the chondrocytes of the proliferative and hypertrophic zones. CONCLUSION: CTGF/Hcs24 expression was detected in both normal and OA chondrocytes of human samples. The results of the current study suggested that expression of CTGF/Hcs24 was concomitant with development of OA lesions and chondrocyte differentiation in chondro-osteophyte.     

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell–specific transmembrane protein (DC‐STAMP)

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF‐κB ligand (GST‐RANKL), and a combination of recombinant CCN2 (rCCN2) and GST‐RANKL significantly enhanced tartrate‐resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST‐RANKL alone. Therefore, we suspected the involvement of CCN2 in cell‐cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real‐time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid‐phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC‐STAMP), which is involved in cell‐cell fusion. The results showed that CCN2 induced and interacted with DC‐STAMP. Furthermore, GST‐RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC‐STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC‐STAMP. © 2011 American Society for Bone and Mineral Research.     

Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells

BACKGROUND: Hepatocyte growth factor (HGF) is a pleiotropic protein with renoprotective functions, which have been attributed at least in part to its ability to counteract the profibrotic effects of transforming growth factor beta (TGF-beta). A major downstream mediator of TGF-beta is connective tissue growth factor (CTGF). However, the molecular mechanisms of CTGF regulation by HGF have not yet been investigated. METHODS: CTGF expression was analysed in human primary tubular epithelial cells (hPTECs) and the cell line HKC-8 by western blotting. Morphological alterations were analysed by immunocytochemistry. RESULTS: HGF induced a transient expression of CTGF, which was maximal after 6 h and returned to baseline after 24 h. Coincubation with TGF-beta increased CTGF protein at 6 h, whereas HGF significantly decreased CTGF induction by TGF-beta after 24 h. Furthermore, HGF induced cell scattering associated with reorganization of focal adhesions and formation of lamellipodia and filopodia. The early induction of CTGF was linked to the HGF-mediated alterations of cell morphology. The PP2 inhibitor of Src-family kinases, which regulate focal adhesion turnover, reduced HGF-mediated upregulation of CTGF. In addition, inhibition of the Rho-kinase, which modulates the actin cytoskeleton, impaired CTGF expression. Combination of both inhibitors further decreased CTGF expression. Comparable inhibitory effects were obtained, when CTGF was induced by the combination of HGF and TGF-beta. CONCLUSIONS: We provide evidence for a dual effect of HGF on CTGF regulation in human tubular epithelial cells: transient upregulation of CTGF in the absence of TGF-beta, which was related to alterations of cell morphology, and interference with TGF-beta-mediated CTGF induction after prolonged incubation.     

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-β-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.     

结缔组织生长因子在椎间盘纤维化和退变中的表达和作用

目的研究疼痛椎间盘组织中结缔组织生长因子（connective tissue growth factor, CTGF）的表达及其在椎间盘纤维化和退变中的作用。方法收集腰椎后路融合过程中切除的43个疼痛的病理椎间盘，来自于28例行腰椎后路椎体间融合手术的严重椎间盘源性下腰痛患者；同时收集16个在MRIT2加权像信号强度明显减弱的无腰痛症状的退变椎间盘，取自于6例腰椎管狭窄症和8例多节段腰椎后路融合的患者（年龄44～75岁，平均53．5岁，男女比例为8：6）和8个正常对照椎间盘，来自于4具新鲜尸体标本（22～39岁，平均28岁）的L。和蛉．椎间盘。均行组织学检查并用免疫组化方法检测CTGF在不同椎间盘组织的表达。结果组织学检查发现，疼痛椎间盘组织显示不同程度的慢性血管化炎症反应。纤维环组织失去正常的胶原纤维板层结构，板层结构断裂、紊乱或相互交叉融合，正常的成纤维细胞被软骨细胞替代。髓核显示明显纤维化、血管浸润或形成炎性肉芽组织，软骨细胞被成纤维细胞所替代。免疫组化染色显示CTGF在疼痛椎间盘大量表达，无腰痛症状的退变椎间盘有少量表达，正常对照椎间盘没有表达。结论疼痛的退变椎间盘在组织学上明显不同于无腰痛症状的退变椎间盘。CTGF在疼痛椎间盘的大量表达可能与椎间盘纤维化和退变过程密切相关。     

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.     

Novel mode of processing and secretion of connective tissue growth factor/ecogenin (CTGF/Hcs24) in chondrocytic HCS-2/8 cells

The synthesis, processing, and secretion of human connective tissue growth factor (CTGF/Hcs24) in a human chondrocytic cell line, HCS-2/8, were analyzed immunochemically. By metabolic pulse-labeling, chasing, and subsequent immunoprecipitation analyses, active synthesis of CTGF was observed not only in growing HCS-2/8 cells, but also in confluent cells. However, secretion and processing of CTGF were found to be regulated differentially, depending upon the growth status. During phases of growth, HCS-2/8 cells released CTGF molecules immediately without sequestering them within the cell layer. In contrast, after the cells reached confluence, the secretion slowed, resulting in an accumulation of CTGF in the cells or extracellular matrices (ECMs). Also, in confluent cell layers, a 10 kDa protein that was reactive to an anti-CTGF serum was observed. This CTGF-related small protein was not detected immediately after labeling, but gradually appeared within 6 h after chase, which suggests its entity as a processed subfragment of CTGF. Surprisingly, the 10 kDa protein was stable even 48 h after synthesis, and was not released by ECM digestion, suggesting an intracellular maintenance and function. Taken together, the behavior of CTGF in HCS-2/8 cells is remarkably different from that reported in fibroblasts, which may represent unique roles for CTGF in the growth and differentiation of chondrocytes.     

Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor).

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage. INTRODUCTION: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2. MATERIALS AND METHODS: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo. RESULTS: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro. CONCLUSION: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.     

结缔组织生长因子促进半月板无血管区损伤愈合

目的：探讨结缔组织生长因子（CTGF）对纤维软骨细胞外基质分泌、VEGF表达及促进半月板无血管区的损伤修复中的作用。方法：将新西兰大白兔半月板白区,经胶原酶消化、离心分离后,提取半月板纤维软骨细胞,培养至第2代。用流式细胞仪鉴定该细胞表面CD31,CD44,CD45和CD105标志物,并用Ⅱ型胶原抗体做免疫细胞化学鉴定,以证明所培养、传代的细胞是纤维软骨细胞。分别将细胞培养在浓度100 ng/ml的CTGF培养基中3、14 d后,通过实时定量聚合酶链反应,检测Ⅰ型胶原、Ⅱ型胶原和VEGF基因的表达变化情况。造模,在兔半月板中央区,制作长3 mm的纵行撕裂。将45只大白兔随机分为3组,处理方式为：半月板单纯缝合术,缝合术加填充PBS-纤维蛋白胶,缝合术加填充1．5滋g CTGF-纤维蛋白胶。术后第1、4、10周用荧光免疫组织化学分析法显示Ⅰ型、Ⅱ型胶原和VEGF在损伤处的表达与分布情况,直观撕裂处的愈合情况。结果：体外实验第14天,定量RT-PCR结果显示,100 ng/ml CTGF组中的Ⅰ型、Ⅱ型胶原和VEGF mRNA表达比PBS对照组,明显增加。体内实验的荧光免疫组织化学结果显示,在术后第10周,CTGF治疗组中的Ⅰ型、Ⅱ型胶原和VEGF已完全填充撕裂损伤处。 PBS-蛋白胶组中,损伤处仍有明显裂隙。结论：CTGF可促进半月板无血管区重要的细胞外基质（Ⅰ型、Ⅱ型胶原）的合成,同时损伤处VEGF的表达活性明显增强,有利于促进无血管区半月板撕裂损伤的愈合。     

Unraveling the role of connective tissue growth factor in diabetic nephropathy

Marked upregulation of the secreted extracellular matrix-associated protein connective tissue growth factor (CTGF) in the glomerulus coincides with the onset of diabetic nephropathy. Recent studies, including the one by Yokoi et al., shed light on the role of CTGF in glomerular injury.