Expression and differential regulation of connective tissue growth factor in pancreatic cancer cells.
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CTGF is an immediate early growth responsive gene that has been shown to be a downstream mediator of TGFbeta actions in fibroblasts and vascular endothelial cells. In the present study hCTGF was isolated as immediate early target gene of EGF/TGFalpha in human pancreatic cancer cells by suppression hybridization. CTGF transcripts were found in 13/15 pancreatic cancer cell lines incubated with 10% serum. In 3/7 pancreatic cancer cell lines EGF/TGFalpha induced a significant rise of CTGF transcript levels peaking 1-2 h after the start of treatment. TGFbeta increased CTGF transcript levels in 2/7 pancreatic cancer cell lines after 4 h of treatment and this elevation was sustained after 24 h. Only treatment with TGFbeta was accompanied by a parallel induction of collagen type I transcription. 15/19 human pancreatic cancer tissues were shown to overexpress high levels of CTGF transcripts. CTGF transcript levels in pancreatic cancer tissues and nude mouse xenograft tumors showed a good correlation to the degree of fibrosis. In situ hybridization and the nude mouse experiments revealed that in pancreatic cancer tissues, fibroblasts are the predominant site of CTGF transcription, whereas the tumor cells appear to contribute to a lesser extent. We conclude that CTGF may be of paramount importance for the development of the characteristic desmoplastic reaction in pancreatic cancer tissues. (+info)
Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.
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Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu. (+info)
Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo.
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Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor alphavbeta3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-alphavbeta3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-alphavbeta3-dependent pathways. (+info)
Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.
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To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR. (+info)
Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo.
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Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process. (+info)
Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis.
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OBJECTIVE: To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP). BACKGROUND: Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis. PATIENTS AND METHODS: CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples. RESULTS: Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues. CONCLUSION: The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP. (+info)
Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3).
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Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha(v)beta(3). Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I(2), which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha(IIb)beta(3)), but not by LM609 (anti-alpha(v)beta(3)), indicating that the interaction is mediated through integrin alpha(IIb)beta(3). In a solid phase binding assay, activated alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alpha(IIb)beta(3) failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin alpha(IIb)beta(3) on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis. (+info)
Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression.
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AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological functions of NOV, as has been reported previously for several signalling peptides and proteins. METHODS: Nuclear and cytoplasmic fractions were prepared from 143 and HeLa cells in which nuclear NOV protein was detected. Western blotting analysis of NOV proteins in both types of fractions was performed using two NOV specific antibodies. Confocal microscopy was used to visualise the nuclear NOV protein in HeLa and 143 cells. A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein. RESULTS: A 31/32 kDa doublet of NOV protein was identified in the nuclear fraction of 143 and HeLa cells. Because the antibodies were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform is probably truncated at the N-terminus and might correspond to the secreted 32 kDa NOV isoform detected in cell culture medium. Confocal microscopy indicated that in addition to the cytoplasmic NOV protein already identified, a nuclear NOV protein was present in both the nucleoplasm and nucleoli of Hela and 143 cells. Screening of cDNA libraries prepared from HeLa cells, Epstein-Barr virus transformed lymphocytes, and normal human brain showed that the NOV protein interacts with the rpb7 subunit of RNA polymerase in a yeast two hybrid system. CONCLUSIONS: The NOV protein detected in the nucleus of 143 and HeLa cells is probably an N-terminus truncated isoform of the secreted 48 kDa NOV protein. A growing body of evidence suggests that novH expression is closely associated with differentiation in normal human tissues and that the nov gene encodes a signalling protein that belongs to an emerging family of cell growth regulators. The nuclear localisation of a NOV isoform potentially provides an additional degree of signalling specificity. The interaction of the NOV protein and the rpb7 subunit of RNA polymerase II in the two hybrid system suggests that NOV might be involved in regulating gene expression at the transcriptional level. As has already been suggested for several other nuclearly located cytokines, the NOV protein does not contain a typical nuclear localisation signal. Therefore, it is possible that it combines with either a receptor or a chaperone during its translocation. Disruption of the balance between the secreted and nuclear NOV isoforms might affect the putative autocrine and paracrine functions of NOV and might be of considerable importance in the development of cancers in which the expression of novH has been shown to be impaired. (+info)