The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor.
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Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology. (+info)
Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP.
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The induction of connective tissue growth factor (CTGF) was investigated in a human renal fibroblast cell line that exhibited many characteristics of primary human renal fibroblasts. Induction of CTGF mRNA was observed after treatment of the cells with transforming growth factor-beta (TGF-beta) or, even more prominently, lysophosphatidic acid (LPA). LPA induced a rapid transient increase in CTGF mRNA expression, with maximal levels being observed after 1 to 2 h. This increase was accompanied by CTGF protein synthesis. Induction of CTGF was insensitive to pertussis toxin and was not dependent on the activation of p42/44 mitogen-activated protein kinases. Inhibition of the proteins of the Rho family with toxin B from Clostridium difficile abrogated basal and LPA-mediated induction of CTGF. Specific targeting of RhoA with C3 exotoxin or of the Rho kinases with the inhibitor Y-27632 similarly prevented induction of CTGF, implicating RhoA as a signaling module downstream of LPA. Inhibition of RhoA depolymerized the actin cytoskeleton, as did treatment with cytochalasin D. Preincubation of the human renal fibroblasts with cytochalasin D prevented induction of CTGF by LPA, indicating a strong contribution of an intact cytoskeleton. Interference with RhoA signaling similarly inhibited the induction of CTGF by TGF-beta. Elevation of intracellular levels of cAMP and thus activation of protein kinase A prevented induction of CTGF expression. The cytoskeletal effects of cAMP, however, were reversed by LPA. These data indicate complex interactions involving LPA-mediated activation of RhoA- and protein kinase A-dependent signaling pathways. The data thus demonstrate the regulatory functions of the small GTPase RhoA and of an intact cytoskeleton in the expression of CTGF after stimulation with LPA or TGF-beta. Analogous signal transduction pathways were previously demonstrated in rat mesangial cells, suggesting a more general role for RhoA in the regulation of CTGF expression. (+info)
Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.
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We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56 kDa) of CTGF in mesangial cell lysates as well as the monomeric (36-38 kDa) and lower molecular mass forms (<30 kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF-V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4 mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30 mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor beta1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover. (+info)
Uptake and intracellular transport of the connective tissue growth factor: a potential mode of action.
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Connective tissue growth factor (CTGF) is a secreted cysteine-rich protein now considered as an important effector molecule in both physiological and pathological processes. An increasing amount of evidence indicates that CTGF plays a key role in the pathogenesis of different fibrotic disorders including diabetic nephropathy. However, the molecular mechanisms by which CTGF exerts its effects are not known. Here we provide the first evidence for the existence of an intracellular transport pathway for the growth factor in human mesangial cells. Our results demonstrate that CTGF is internalized from the cell surface in endosomes and accumulates in a juxtanuclear organelle from which the growth factor is then translocated into the cytosol. In the cytosol CTGF is phosphorylated by protein kinase C and PMA treatment can enhance this phosphorylation. Phosphorylated CTGF may have an important role in the cytosol, but it is also translocated into the nucleus where it may directly affect transcription. (+info)
Lefty contributes to the remodeling of extracellular matrix by inhibition of connective tissue growth factor and collagen mRNA expression and increased proteolytic activity in a fibrosarcoma model.
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Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo. (+info)
Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus.
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AIMS: To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta1 (TGF-beta1) in the mouse uterus. METHODS: In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) and TGF-beta1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or (2) ovariectomised (OVX) and administered estradiol-17beta (E2) or progesterone (P4), either alone or in combination. Uteri collected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-beta1. RESULTS: On days 0.5-2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-beta1 were principally colocalised to uterine epithelial cells, with much smaller amounts in the stroma. On days 3.5-4.5, there was a reduction of CCN2 (CTGF) and TGF-beta1 in the epithelium but an increase in stromal and endothelial cells, corresponding to a period of extracellular matrix remodelling and neovascularisation within the endometrium. In OVX mice, epithelial cells were weakly positive for both CCN2 (CTGF) and TGF-beta1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently stimulated in OVX mice cells by E2. These effects were antagonised by P4, which itself transiently stimulated epithelial CCN2 (CTGF) production, although less robustly than E2. CTGF and TGF-beta1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the stroma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-beta1 were lower after co-administration of E2 and P4 than in response to each hormone individually. Although ccn2 (ctgf) is a TGF-beta1 inducible gene in other systems, and both growth factors were often co-localised in uterine tissues in these studies, several treatment regimens resulted in high amounts of TGF-beta1 protein in stromal cells without the concomitant production of ccn2 (ctgf) mRNA. CONCLUSIONS: Maternal factors are principal cues for CCN2 (CTGF) and TGF-beta1 production in the uterus because (1) their expression during pseudopregnancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-beta dependent and independent mechanisms of ccn2 (ctgf) gene transcription exist in the uterus that are variably regulated by steroid hormones. Collectively, the data support a role for CCN2 (CTGF) in mediating the effects of steroid hormones and TGF-beta on endometrial function. (+info)
PURPOSE: Transforming growth factor beta1 (TGF-beta) stimulates the differentiation of myofibroblasts as indicated by the nascent expression of alpha-smooth muscle (alpha-SM) actin protein and its organization into stress fibers. Downstream messengers of TGF-beta in the conversion from the fibroblast to the myofibroblast phenotype were investigated. Whether TGF-beta increases the transcription of a second growth factor, connective tissue growth factor 1 (CTGF), which could mediate myofibroblast differentiation, was evaluated. CTGF, a newly identified growth factor, is highly expressed in dermal granulation tissue. METHODS: In this study, primary cultures of rabbit corneal fibroblasts were exposed to growth factors to investigate CTGF mRNA and protein expression during myofibroblast differentiation. Statistical analysis was used to evaluate the impact of growth factor treatment on myofibroblast differentiation. RESULTS: . TGF-beta treatment induced both CTGF mRNA and protein in rabbit corneal fibroblasts; in contrast, fibroblast growth factor-2 (FGF) and heparin led to a decrease in CTGF mRNA. Addition of recombinant CTGF to rabbit corneal fibroblast cultures did not significantly increase alpha-SM actin mRNA or protein nor did it appear to affect assembly of alpha-SM actin stress fibers. CONCLUSIONS: This is the first study to present evidence for the induction of CTGF by TGF-beta treatment of corneal fibroblasts. It is doubtful that CTGF is the TGF-beta mediator of the corneal fibroblast to myofibroblast transition because CTGF does not induce alpha-SM actin in subconfluent fibroblast cultures. CTGF may play a supporting role in myofibroblast differentiation. (+info)
Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.
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Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 +/- 2.0 mg versus 17.1 +/- 1.4 mg, P < 0.01) was preceded by significant elevations in alpha1(I) procollagen and CTGF mRNA levels (3.0 +/- 0.4-fold and 6.3 +/- 0.4-fold respectively, (P < 0.01), and total inflammatory cell number. UK-156406, administered at an anticoagulant dose, attenuated lung collagen accumulation in response to bleomycin by 35 +/- 12% (P < 0.05), inhibited alpha1(I) procollagen and CTGF mRNA levels by 50% and 35%, respectively (P < 0.05), but had no effect on inflammatory cell recruitment. This is the first report showing that direct thrombin inhibition abrogates lung collagen accumulation in bleomycin-induced pulmonary fibrosis. (+info)