Regulation of connective tissue growth factor (ccn2; ctgf) gene expression in human mesangial cells: modulation by HMG CoA reductase inhibitors (statins).

AIM: Connective tissue growth factor (ccn; ctgf) gene expression is upregulated in fibrotic renal glomeruli. Therefore, the regulation and pharmacological modulation of ccn2 (ctgf) mRNA expression was investigated in a human renal mesangial cell line. METHODS: A human renal mesangial cell line was cultured in vitro under standard conditions. After stimulation, RNA was extracted and ccn2 (ctgf) mRNA expression assessed by northern blot analysis. RESULTS: The expression of ccn2 (ctgf) mRNA was transiently upregulated by fetal calf serum. Very rapid onset but short lasting ccn2 (ctgf) mRNA expression was observed after stimulation with lysophosphatidic acid, a bioactive lipid, which activates G protein coupled receptors. Induction of ccn2 (ctgf) mRNA expression by transforming growth factor beta (TGF-beta) was more prolonged and lasted for more than one day. The small GTPases of the Rho family were essential for basal as well as induced ccn2 (ctgf) expression: preincubation of the cells with toxin B from Clostridium difficile abrogated ccn2 (ctgf) mRNA expression. HMG CoA reductase inhibitors, which are therapeutically used as lipid lowering drugs, interfere with the isoprenylation and thus activation of Rho proteins. Simvastatin, an HMG CoA reductase inhibitor, inhibited ccn2 (ctgf) mRNA expression in a concentration dependent manner (IC(50): 1-2 microM). CONCLUSION: Statins were identified as potent inhibitors of ccn2 (ctgf) mRNA expression in mesangial cells, and therefore might be of potential use to modulate the excessive ccn2 (ctgf) expression in mesangial cells related to glomerular fibrosis.     

The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis

Connective tissue growth factor (CTGF) is a 38 kDa, cysteine rich, extracellular matrix protein composed of 4 domains or modules. CTGF has been shown to regulate a diverse array of cellular functions and has been implicated in more complex biological processes such as angiogenesis, chondrogenesis, and osteogenesis. A role for CTGF in the development and maintenance of skeletal tissues first came to light in studies demonstrating its expression in cartilage and bone cells, which was dramatically increased during skeletal repair or regeneration. The physiological significance of CTGF in skeletogenesis was confirmed in CTGF-null mice, which exhibited multiple skeletal dysmorphisms as a result of impaired growth plate chondrogenesis, angiogenesis, and bone formation/mineralization. Given the emerging importance of CTGF in osteogenesis and chondrogenesis, this review will focus on its expression in skeletal tissues, its effects on osteoblast and chondrocyte differentiation and function, and the skeletal implications of ablation or over-expression of CTGF in knockout or transgenic mouse models, respectively. In addition, this review will examine the role of integrin-mediated signaling and the regulation of CTGF expression as it relates to skeletogenesis. We will emphasize CTGF studies in bone or bone cells, and will identify opportunities for future investigations concerning CTGF and chondrogenesis/osteogenesis.     

Regulation of pancreatic inflammation by connective tissue growth factor (CTGF/CCN2)

Pancreatitis is caused by long‐term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells (PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin‐1β (IL‐1β), chemokine (C‐C motif) ligand 3 (CCL3), or connective tissue growth factor (CTGF/CCN2) by Day 16 and this was associated with increased infiltration of F4/80‐positive macrophages and increased expression of pancreatic CTGF/CCN2 mRNA. Compared with wild‐type Swiss Webster mice, ethanol treatment of pan‐green fluorescent protein (GFP)‐CTGF/CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/CCN2 and a significant increase in pancreatic infiltration of F4/80‐positive macrophages or NIMP‐R14‐positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/CCN2 resulted in enhanced expression of IL‐1β or CCL3. Conditioned medium from CTGF/CCN2‐treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose‐dependent manner by addition of BX471, an inhibitor of chemokine (C‐C motif) receptor 1. These results reveal that acinar CTGF/CCN2 plays a novel role in alcohol‐induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL‐1β or CCL3. The early production of CTGF/CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury.     

Pulmonary hypoplasia in the connective tissue growth factor (Ctgf) null mouse.

Connective tissue growth factor (CTGF) is a mediator of growth factor activity, and Ctgf knockouts die at birth from respiratory failure due to skeletal dysplasia. Previous microarray analysis revealed Ctgf down-regulation in the hypoplastic lungs of amyogenic mouse embryos. This study, therefore, examined pulmonary development in Ctgf-/- mouse fetuses to investigate if respiration could also have been impaired by lung abnormalities. The Ctgf-/- lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. PDGF-B, its receptor and IGF-I, were markedly attenuated and the TTF-1 gradient lost. Type II pneumocyte differentiation was perturbed, the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant-associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. Our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia by both disrupting basic lung developmental processes and restricting thoracic expansion.     

In situ expression of connective tissue growth factor in human crescentic glomerulonephritis

Connective tissue growth factor (CTGF) has recently been recognized as an important profibrotic factor and is up-regulated in various renal diseases with fibrosis. The present study describes the sequential localization of CTGF mRNA and its association with transforming growth factor (TGF)-1 in human crescentic glomerulonephritis (CRGN). Furthermore, we examined the phenotype of CTGF-expressing cells using serial section analysis. Kidney biopsy specimens from 18 CRGN patients were examined using in situ hybridization and immunohistochemistry. CTGF mRNA was expressed in the podocytes and parietal epithelial cells (PECs) in unaffected glomeruli. In addition, it was strongly expressed in the cellular and fibrocellular crescents, particularly in pseudotubule structures. Serial sections revealed that the majority of CTGF mRNA-positive cells in the crescents co-expressed the epithelial marker cytokeratin, but not a marker for macrophages. Moreover, TGF-1, its receptor TGF- receptor-I, and extracellular matrix molecules (collagen type I and fibronectin) were co-localized with CTGF mRNA-positive crescents. Our results suggest that CTGF is involved in extracellular matrix production in PECs and that it is one of the mediators promoting the scarring process in glomerular crescents.     

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G‐415 cells, and the effects on cell viability, anchorage‐independent growth and migration was assessed by comparing them to scrambled vector‐transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage‐independent growth (P < 0.05). An increased p27 expression was observed in G‐415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells.     

结缔组织生长因子促纤维化作用及其表达调节的研究进展

Connective tissue growth factor (CTGF) has recently received much attention as a possible key determinant of progressive fibrosis. It promotes tissue fibrosis through different pathways, such as cell proliferation, extracellular matrix accumulation and cell transdifferentiation. A number of regulators of CTGF expression have been identified, including transformiing growth factor β, vascular endothelial growth factor, tumor necrosis factor α, etc. The mechanism of profibrotic effect by CI‘GF was reviewed.     

Gene expression of connective tissue growth factor in adult mouse

The connective tissue growth factor (CTGF) is a well-known fibroblast mitogen and angiogenic factor that plays an important role in bone formation during embryogenesis. In the adult, CTGF is involved in wound healing as well as fibrotic and vascular disease. However, little is known about its physiological functions under non-pathological conditions in the adult organism. Here, we describe the cellular site of the CTGF mRNA expression in adult male and female mice as revealed by in situ hybridization histochemistry. Strong and persistent CTGF gene expression was particularly prominent in the mesenchyme of the cardiovascular system (aorta, auricular tissue, renal glomeruli), the mesenchyme surrounding the ovarian follicles or the testicular tubes in the gonadal tissue, and the subcapsular mesenchyme bordering densely innervated parts of whisker hair vibrissae. CTGF hybridization signals were not observed in the mesenchyme of many other organs including gut, muscle, liver or most parts of the lymphatic tissue. Strong expression was also present in the primary (early) ovarian follicles, the epithelium of the deep uterine glands and on myenteric ganglia neurons. These data suggest a selective and continuous mesenchymal function in the gonads and those tissues attracting very strong vascular supply or peripheral innervation. CTGF may also be involved in the cyclical proliferation of the uterine gland epithelium and in the early stages of follicular maturation, as well as in the neuropeptide regulation in the gut, cardiovascular and renal systems.     

Modulation of the expression of connective tissue growth factor (CTGF) by alterations of the cytoskeleton

Introduction In closing wounds or developing fibrotic tissue, fibroblasts are exposed to mechanical stress, which leads to alterations in cell morphology and reorganization of the cytoskeleton. The impact of morphological changes on gene expression was investigated in the present study. As a model system, we used a human renal fibroblast cell line and studied the expression of connective tissue growth factor (CTGF). CTGF is a downstream mediator of TGF‐β mediating many of the pro‐fibrotic actions of this growth factor and has also been shown to be up‐regulated by static or dynamic pressure. The molecular mechanisms regulating CTGF expression related to stress are not yet known. Materials and methods A human renal fibroblast cell line was kindly provided by Dr Müller, Göttingen, Germany. These cells express CTGF upon treatment with soluble stimuli ( Heusinger‐Ribeiro et al. 2001 ; Eberlein et al. 2001 ). CTGF expression was detected by northern and Western blot analysis. The actin cytoskeleton was visualized by rhodamine phalloidin staining, and microtubules were detected by immunocytochemistry. Results Low concentrations of the microtubule disrupting agents nocodazole and colchicine strongly up‐regulated CTGF mRNA and protein expression in the human renal fibroblast cell line TK173. The up‐regulation was prevented by stabilization of the microtubules by paclitaxel (taxol). As a consequence of microtubule disruption, the small GTPase RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction: interference with RhoA signalling by simvastatin, toxin B and Y27632 prevented up‐regulation of CTGF. The important role of RhoA was supported by an increased CTGF expression upon overexpression of constitutively active RhoA. Direct disassembly of the actin cytoskeleton by latrunculin B interfered with colchicine‐mediated up‐regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D unexpectedly increased CTGF expression. This indicated that the content of F‐actin per se was not the major determinant for CTGF gene expression. It has been shown, however, that cytochalasin D sequesters G‐actin, whereas latrunculin increases the level of G‐actin. Our data are thus in accordance with an inverse correlation between G‐actin levels and CTGF expression. Discussion These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF. Recently, decreased levels of G‐actin were observed in vascular smooth muscle cells in response to increased vascular pressure ( Cipolla et al. 2002 ). Our findings thus provide a molecular basis for the observation that CTGF is up‐regulated in cells exposed to mechanical stress.