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.     

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.     

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.     

Collaborative action of M-CSF and CTGF/CCN2 in articular chondrocytes: possible regenerative roles in articular cartilage metabolism

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo.     

Aldosterone induces CTGF in mesangial cells by activation of the glucocorticoid receptor.

BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.     

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.     

CTGF expression in mesangial cells: involvement of SMADs,MAP kinase,and PKC

BACKGROUND: The induction of excess matrix in renal fibrosis seems to be mediated, at least in part, by the transforming growth factor-beta (TGF-beta)-mediated induction of connective tissue growth factor (CTGF) in mesangial cells. METHODS: By examining CTGF protein and mRNA expression and promoter activity in the presence or absence of TGF-beta or inhibitors, the signaling pathways controlling basal and TGF-beta-induced CTGF expression in mesangial cells were investigated. RESULTS: TGF-beta enhances CTGF mRNA and protein expression in mesangial cells. Mutation of a consensus SMAD binding element in the CTGF promoter completely abolished TGF-beta-induced CTGF expression and reduced basal CTGF expression. The previously identified basal control element-1 (BCE-1) site, but not Sp1 contributes to basal CTGF promoter activity. Ras/MEK/ERK, protein kinase C (PKC) and tyrosine kinase activity also contribute to basal and TGF-beta-induced CTGF promoter activity in cultured mesangial cells. CONCLUSIONS: The TGF-beta-induction of CTGF in mesangial cells requires SMADs and PKC/ras/MEK/ERK pathways. SMADs are involved in basal CTGF expression, which presumably reflects the fact that mesangial cells express TGF-beta endogenously. TGF-beta also induces CTGF through ras/MEK/ERK. Inhibiting ras/MEK/ERK seems not to reduce phosphorylation (that is, activation) of SMADs, suggesting that SMADs, although necessary, are insufficient for the TGF-beta-stimulation of the CTGF promoter through ras/MEK/ERK. Thus, maximal TGF-beta induction of CTGF requires synergy between SMAD and ras/MEK/ERK signaling.     

结缔组织生长因子基因启动子多态性与IgA肾病的关系

目的 研究结缔组织生长因子基因启动子多态性及其与上海地区中国人IgA肾病发生的相关性。方法 应用聚合酶链反应(PCR)-PCR产物直接测序技术，对50例IgA肾病(IgAN)患者和30例健康对照者(包括28例中国人和2例白种人)进行结缔组织生长因子基因ccn2(ctgf)启动子多态性研究。结果 检测到1种多态和1种突变。多态性改变G-447／C位于ccn2基因启动子的骨髓锌指蛋白(MZF)1结合基序附近。50例IgAN患者中有G．447／C多态性2例(4％)；28例中国健康对照者中有1例(3、57％)，该多态性频率两组间差异无显著性意义。在1例IgAN患者中发现ccn2基因1号外显子第20位核苷酸处存在G→T颠换，该突变接近ccn2基因mRNA的5’端帽式结构区，30例正常人中未发现这种突变。结论 (1)中国人群中有ccn2基因启动子多态性存在，ccn2基因启动子G-447／C多态性可能与IgAN发生无直接相关。(2)IgAN患者中存在ccn2基因1号外显子5’非编码区G→T颠换。经检索，该突变属首次报道。     

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.     

Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression

Connective tissue growth factor (CTGF) is a 38 Kda cysteine-rich, heparin-binding peptide that has been implicated in several normal and abnormal physiological processes. CTGF has been shown to be induced by transforming growth factor-beta. Previous studies in our pig model of skin wound healing showed a coordinate expression of transforming growth factor-beta and CTGF during the healing process. To better understand the function of CTGF during wound healing, normal porcine fibroblasts were isolated from skin samples from SPF Yorkshire pigs. At fourth passage the cells were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum and at 80% confluence the medium was replaced with supplemented serum-free medium. After a further 24 hours, cells were treated with 0, 10, 25, 50, 100, and 500 ng/ml of 38 Kda or 16-20 Kda (C-terminal truncated form) recombinant expressed human CTGF for 24 hours or treated with 100 ng/ml for 0, 12, 24, and 48 hours. Subsequently, CTGF effects on cell DNA synthesis and mRNA levels for a subset of relevant molecules were assessed. The results showed that in cells treated with 38 Kda rhCTGF, mRNA levels for types I and III collagen, fibromodulin, and basic fibroblast growth factor were significantly up-regulated, but mRNA levels for HSP47, decorin, biglycan, and versican were not significantly altered. mRNA levels for CTGF were also significantly increased, indicating autoregulation of expression. However, mRNA levels for transforming growth factor-beta, inteleukins 1 and 6, tumor necrosis factor-alpha, and nerve growth factor did not change. Interestingly, mRNA levels for the tissue inhibitors of metalloproteinase-1, -2, -3 and -4 were observed to significantly increase, but in contrast, mRNA levels for matrix metalloproteinases-1, -2, -9 were not significantly altered by exposure of the cells to the 38 Kda form of CTGF. In addition, DNA synthesis was augmented in the presence of 38 Kda rhCTGF. However, the truncated 16-20 Kda form of rhCTGF appeared to have none of these effects on porcine fibroblasts. These results indicate that in order to induce changes in porcine fibroblasts a molecule with an intact C-terminal domain is required, and that CTGF regulates porcine fibroblast extracellular matrix molecule, growth factor, and proteinase inhibitor gene expression without apparently affecting matrix metalloproteinase mRNA levels. These findings suggest that CTGF contributes to the anabolic environment during skin wound healing via selective modulation of fibroblast proliferation and changes to gene expression.     

转化生长因子β1对肾小管上皮细胞中结缔组织生长因子基因启动子活性的影响

目的探讨转化生长因子[β1（TCF-β1）对人近端肾小管上皮细胞系HK-2中结缔组织生长因子（CTGF）基因启动子活性的调控作用，以及丝裂原激活蛋白激酶（MAPK）途径对该生长因子作用的影响。方法构建含有人类CTGF基因启动子的报告基因pCTGF-luc，将其瞬时转染HK-2细胞。通过检测荧光素酶的活性观察TGF-β1和MAPK途径抑制剂对CTGF基因启动子活性的影响。结果TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子的活性。最佳刺激浓度是5ng／ml，最佳刺激时间为12h，荧光素酶相对活性分别为对照组的1．82倍和2．10倍（P〈0．05）。应用PD98059、SB203580和SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶（ERK）、蛋白激酶p38（p38MAPK）和c-Jun-氨基末端激酶（JNK）通路，对TGF-β1上调CTGF启动子活性的作用有不同影响。PD98059显著增加HK-2中pCTGF-luc的基础活性．并在一定浓度范围内（0．5～10μmol／L）促进TGF-β1的上调作用。SB203580对pCTGF-luc基础活性无影响，但以剂量依赖方式显著抑制TGF-β1的激活效应。而SP600125对基础状态和TGF-β1刺激下CTGF基因启动子活性无影响。结论TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子活性，在转录水平调节CTGF表达。MAPK途径的ERK和p38MAPK通路可影响TGF-β1的这一调控作用。     

The effect of connective tissue growth factor on renal fibrosis and podocyte injury in hypertensive rats

Objective: The aim of this study was to evaluate the roles of podocalyxin (PCX) and connective tissue growth factor (CTGF) in spontaneously hypertensive rats. Methods: Spontaneously hypertensive rats (SHR) and normotensive control Wistar–Kyoto (WKY) rats were divided into groups referred to as SHR 12W, SHR 24W, WKY 12W and WKY 24W. Systolic blood pressure and 24-hour total uric protein were measured every two weeks in the respective groups. CTGF, PCX, alpha-smooth muscle actin (α-SMA) and collagen-III were evaluated via immunohistochemical staining. In addition, CTGF, PCX, and α-SMA gene expression levels were determined by analyzing mRNA levels. Results: More kidney lesions occurred alongside foot processes effacement in SHR 24W rats than in SHR 12W rats. In SHR 12W rats, blood pressure and 24-hour total uric protein level were elevated and continued to increase thereafter. In the SHR 12W and SHR 24W groups, the expression of CTGF, α-SMA and collagen-III was significantly increased. Immunohistochemical staining showed that PCX expression was significantly reduced in the SHR group and CTGF expression was increased. A significant decrease in PCX mRNA and an increase in CTGF mRNA were observed in SHR 24W rats relative to SHR 12W rats. Conclusion: Both the overexpression of CTGF and the loss of podocalyxin reflect renal damage in spontaneously hypertensive rats. CTGF and PCX may be involved in the mechanisms of podocyte injury and apoptosis induced by hypertension.     

Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas

OBJECT: The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the cytotoxic effect of alkylating agents on tumor cells. The presence of two nonconsensus glucocorticoid-responsive elements in the human MGMT promoter region indicates the potential regulation of MGMT expression by glucocorticoid agents. This study was performed to elucidate whether dexamethasone affects the expression of MGMT in glioblastoma multiforme (GBM) cells, thereby limiting the benefit of chemotherapeutic alkylating agents. METHODS: Four GBM cell lines (A172, T98G, U138MG, and U87MG) were exposed to the alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) with or without dexamethasone. The expression levels of MGMT were correlated with the cytotoxic effects of ACNU in GBM cells. In the presence of ACNU alone, dexamethasone alone, and the combination of both agents, messenger RNA expression of MGMT was induced to varying degrees with the highest increases seen in the later conditions. This dexamethasone-dependent induction of the MGMT gene was even observed in U87MG cells in which the promoter is methylated, although the absolute expression of MGMT mRNA was the lowest in that cell line. The induction of MGMT by dexamethasone was associated with an increased resistance of these cells to ACNU. CONCLUSIONS: These results indicate that dexamethasone-mediated upregulation of MGMT limits the efficiency of alkylating agents in the treatment of malignant gliomas.     

Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-β1 where it acts as a downstream mediator of TGF-β1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-β1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-β1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-β1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-β1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-β1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-β1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-β1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-β in some cell types, we determined its role in TGF-β1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-β1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-β1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-β1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (− 787 to + 1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-β1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-β1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-β1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-β1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.