CTGF/Hcs24 interacts with the cytoskeletal protein actin in chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) displays multiple functions in several types of mesenchymal cells, including the promotion of proliferation and differentiation of chondrocytes. Recently, the internalization and intracellular function of CTGF/Hcs24 were indicated as well. In this study, a binding protein for this factor was purified from the cytosolic fraction of human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by CTGF/Hcs24-affinity chromatography. The apparent molecular weight of the protein was 42kDa and determination of the internal amino acid sequence revealed this protein to be beta- or gamma-actin. An in vitro competitive binding assay of 125I-labeled recombinant CTGF/Hcs24 with cold-rCTGF/Hcs24 showed that the binding between actin and 125I-CTGF/Hcs24 was specific. Immunoprecipitation analysis also showed that CTGF/Hcs24 bound to actin in HCS-2/8 cells. However, rCTGF/Hcs24 had no effects on the expression level of gamma-actin mRNA or total actin protein. These findings suggest that a significant portion of intracellular CTGF/Hcs24 may regulate certain cell biological events in chondrocytes through the interaction with this particular cytoskeletal protein.     

Calcium induces differentiation of primary human salivary acinar cells

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.     

Role of CTGF/HCS24/ecogenin in skeletal growth control

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions.     

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130-140; Shimo et al., 1999, J Biochem 126:137-145; Nakanishi et al., 2000, Endocrinology 141:264-273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using (125)I-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of (125)I-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of (125)I-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called "ecogenin: endochondral ossification genetic factor." Copyright 2000 Wiley-Liss, Inc.     

Conserved repressive regulation of connective tissue growth factor/hypertrophic chondrocyte-specific gene 24 (ctgf/hcs24) enabled by different elements and factors among vertebrate species

CTGF/Hcs24 is a multifunctional growth factor that potentiates the growth and differentiation of various cells. Our previous study revealed that the 3'-UTR of mammalian CTGF/Hcs24 mRNA contains a small segment that represses the gene expression in cis fashion. In this study, we isolated and characterized a chicken CTGF/Hcs24 cDNA clone. Chicken ctgf/hcs24 mRNA showed highly conserved homology in the ORF to that of mammalian species, whereas the homology in the 3'-UTR was relatively low. Northern blotting analysis revealed that chicken ctgf/hcs24 mRNA was expressed most strongly in cartilage, and also in brain, lung, heart, but faintly in liver. Thereafter we analyzed the functional potential of the 3'-UTR of ctgf/hcs24 cDNA to regulate its gene expression by reporter gene assay, and found that it repressed gene expression in cis fashion, specifically in avian cells, but not in mammalian cells. Conversely, the mammalian 3'-UTR showed less repressive activity in avian cells than in mammalian cells. Deletion analysis showed that a segment near the polyadenyl tail of the 3'-UTR of chicken ctgf/hcs24 played an important functional role, unlike in the mammalian species. Thus, we uncovered a novel mode of functional conservation of the ctgf/hcs24 3'-UTR among vertebrate species mediated by different factors.     

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.     

Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): detection of HTLV-I tax-induced CTGF from a human carcinoma cell line

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.     

Lung tumor growth-promoting function of peroxiredoxin 6

This study compared lung tumor growth in PRDX6-overexpressing transgenic (Tg) mice and normal mice. These mice expressed elevated levels of PRDX6 mRNA and protein in multiple tissues. In vivo, Tg mice displayed a greater increase in the growth of lung tumor compared with normal mice. Glutathione peroxidase and calcium-independent phospholipase 2 (iPLA2) activities in tumor tissues of Tg mice were much higher than in tumor tissues of normal mice. Higher tumor growth in PRDX6-overexpressing Tg mice was associated with an increase in activating protein-1 (AP-1) DNA-binding activity. Moreover, expression of proliferating cell nuclear antigen, Ki67, vascular endothelial growth factor, c-Jun, c-Fos, metalloproteinase-9, cyclin-dependent kinases, and cyclins was much higher in the tumor tissues of PRDX6-overexpressing Tg mice than in tumor tissues of normal mice. However, the expression of apoptotic regulatory proteins including caspase-3 and Bax was slightly less in the tumor tissues of normal mice. In tumor tissues of PRDX6-overexpressing Tg mice, activation of mitogen-activated protein kinases (MAPKs) was much higher than in normal mice. In cultured lung cancer cells, PRDX6 siRNA suppressed glutathione peroxidase and iPLA2 activities and cancer cell growth, but the enforced overexpression of PRDX6 increased cancer cell growth associated with their increased activities. In vitro, among the tested MAPK inhibitors, c-Jun NH₂-terminal kinase (JNK) inhibitor clearly suppressed the growth of lung cancer cells and AP-1 DNA binding, glutathione peroxidase activity, and iPLA2 activity in normal and PRDX6-overexpressing lung cancer cells. These data indicate that overexpression of PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities through the upregulation of the AP-1 and JNK pathways.     

Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose

Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of Ctgf mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.     

骨骼肌特异性敲除TβRⅡ小鼠模型的建立

目的:建立骨骼肌特异性敲除转化生长因子受体Ⅱ(TβRⅡ)小鼠模型,为进一步研究TβRⅡ在骨骼肌发育和分化中的作用奠定基础。方法:首先将TβRⅡflox/flox转基因小鼠与上游携带肌酸激酶(MCK)启动子的MCK-Cre转基因小鼠进行杂交,培育繁殖出TβRⅡflox/wt/MCK-Cre(+)双转基因小鼠。然后利用TβRⅡflox/wt/MCK-Cre(+)双转基因小鼠与TβRⅡflox/flox转基因小鼠进行杂交,繁殖培育出在骨骼肌内特异敲除TβRⅡ基因的TβRⅡflox/flox/MCK-Cre(+)小鼠。结果:利用Cre/loxP技术世界上首次成功繁殖培育出有活力的且发育正常的TβRⅡ基因敲除小鼠。     

Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP.

Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.