Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis.
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Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-beta1) and thus a potential target for antifibrotic treatment strategies. CTGF is up-regulated in disorders such as atherosclerosis, scleroderma, and fibrosis of kidneys and lungs. We investigated the temporospatial expression patterns of CTGF and TGF-beta1 mRNA in rat livers with acute fibrogenesis (after a single dose of CCl(4)) and with advanced fibrosis (6 weeks after complete bile duct occlusion). Multiprobe ribonuclease protection assay revealed increasing TGF-beta1 and CTGF mRNA levels 6 hours after injection of CCl(4), with peak levels after 72 hours. In biliary fibrosis TGF-beta1 and CTGF mRNA levels increased fourfold and sevenfold, respectively (P: < 0.001). In situ hybridization combined with cell-specific markers revealed CTGF transcripts in desmin-positive cells after a single dose of carbon tetrachloride, whereas no transcripts were found in normal livers. In biliary fibrosis, however, proliferating bile duct epithelial cells were the predominant source of CTGF mRNA. We conclude that in rat liver fibrogenesis CTGF is up-regulated in close association with TGF-beta1 and that, contrary to a previous report, not solely hepatic stellate cells but activated bile duct epithelial cells are the main source of this profibrogenic factor. (+info)
NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
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The CCN family of genes presently consists of six distinct members encoding proteins that participate in fundamental biological processes such as cell proliferation, attachment, migration, differentiation, wound healing, angiogenesis, and several pathologies including fibrosis and tumorigenesis. Whereas CYR61 and CTGF were reported to act as positive regulators of cell growth, NOV (nephroblastoma overexpressed) provided the first example of a CCN protein with negative regulatory properties and the first example of aberrant expression being associated with tumour development. The subsequent discovery of the ELM1, rCOP1, and WISP proteins has broadened the variety of functions attributed to the CCN proteins and has extended previous observations to other biological systems. This review discusses fundamental questions regarding the regulation of CCN gene expression in normal and pathological conditions, and the structural basis for their specific biological activity. After discussing the role of nov and other CCN proteins in the development of a variety of different tissues such as kidney, nervous system, muscle, cartilage, and bone, the altered expression of the CCN proteins in various pathologies is discussed, with an emphasis on the altered expression of nov in many different tumour types such as Wilms's tumour, renal cell carcinomas, prostate carcinomas, osteosarcomas, chondrosarcomas, adrenocortical carcinomas, and neuroblastomas. The possible use of nov as a tool for molecular medicine is also discussed. The variety of biological functions attributed to the CCN proteins has led to the proposal of a model in which physical interactions between the amino and carboxy portions of the CCN proteins modulate their biological activity and ensure a proper balance of positive and negative signals through interactions with other partners. In this model, disruption of the secondary structure of the CCN proteins induced by deletions of either terminus is expected to confer on the truncated polypeptide constitutive positive or negative activities. (+info)
Differential expression of multiple genes during articular chondrocyte redifferentiation.
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Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10(6) cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10(4) cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation. (+info)
Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1.
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Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation. (+info)
Regulation of connective tissue growth factor (ccn2; ctgf) gene expression in human mesangial cells: modulation by HMG CoA reductase inhibitors (statins).
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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. (+info)
The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts.
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Although the role of transforming growth factor beta (TGFbeta) in initiating fibrosis is well established, the role that TGFbeta plays in maintaining fibrosis is unclear. The gene encoding connective tissue growth factor (ccn2; ctgf), which promotes fibrosis, is not normally expressed in dermal fibroblasts unless TGFbeta is present. However, in dermal fibroblasts cultured from lesional areas of scleroderma, ccn2 (ctgf) is expressed constitutively. The contribution of several elements in the ccn2 (ctgf) promoter to basal and TGFbeta induced ccn2 (ctgf) expression in normal and scleroderma fibroblasts has been investigated. A functional SMAD binding site in the ccn2 (ctgf) promoter that is necessary for the TGFbeta mediated induction of this gene has been identified. The previously termed TGFbeta responsive enhancer (TGFbetaRE) in the ccn2 (ctgf) promoter has been found to be necessary for basal promoter activity in normal fibroblasts. The SMAD element is not necessary for the high ccn2 (ctgf) promoter activity seen in scleroderma fibroblasts. However, mutation of the previously termed TGFbetaRE reduces ccn2 (ctgf) promoter activity in scleroderma fibroblasts to that seen in normal fibroblasts. Thus, the maintenance of the scleroderma phenotype, as assessed by a high degree of ccn2 (ctgf) promoter activity, appears to be relatively independent of SMAD action and seems to reflect increased basal promoter activity. (+info)
The expression of ccn3 (nov) RNA and protein in the rat central nervous system is developmentally regulated.
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AIMS: To establish the expression pattern of ccn3 (nov) in the central nervous system (CNS) of adult rats and to determine whether spatiotemporal variations in the expression of ccn3 (nov) are related to specific developmental stages and/or specific CNS functions. METHODS: The sites of ccn3 (nov) expression have been identified by in situ hybridisation using didoxigenin labelled cRNA and by the reverse transcription-polymerase chain reaction (RT-PCR). The rat CCN3 (NOV) protein was characterised by western blotting performed on brain extracts. The localisation of the CCN3 (NOV) protein in the brain was established by immunocytochemistry. RESULTS: Increased expression of ccn3 (nov) was detected in the developing brain of rats after birth, as shown by RT-PCR and immunocytochemistry analysis performed on a series of samples taken between day 5 (P5) and day 300 (P300), with a pronounced peak between P15 and P150, suggesting that CCN3 (NOV) might play a role in the maintenance or establishment of specific brain functions. The relatively high amounts of an N-terminal truncated CCN3 (NOV) related protein detected both in the brain tissues and cerebrospinal fluid suggested that post translational processing of CCN3 (NOV) might be particularly prevalent in the brain. Such processing might be of biological importance in the light of the previously reported growth stimulatory effects of N-terminal truncated CCN3 (NOV) isoforms. CONCLUSIONS: The postnatal differential expression of ccn3 (nov) in the brain of developing rats suggests that CCN3 (NOV) might be involved in the acquisition of specific functions. The rat species provides an as yet unequalled system for these studies. Because the CCN3 (NOV) protein is detected in restricted areas of the brain, it will be interesting to establish whether variations of ccn3 (nov) expression are associated with normal cognitive processes and whether ccn3 (nov) expression is affected by aging. In addition, because CCN3 (NOV) is found in the spinal cord and along the axonal processes, it will be of interest to determine the expression of the normal and truncated isoforms of CCN3 (NOV) in various pathological conditions, such as neurodegenerative diseases. (+info)
Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms.
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BACKGROUND: Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor beta1 (TGF-beta1), interleukin 10 (IL-10), and tumour necrosis factor alpha (TNF-alpha), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. AIM: To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. MATERIALS/METHODS: Seventy seven human DNA samples were sequenced-45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)-using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. RESULTS: A C to G substitution was identified at position -132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position -447. These substitutions at -132 and -447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position -43 (G), -47 (C), -71 (G) and a C to T substitution at position -198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. CONCLUSION: Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements. (+info)