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Connective Tissue Growth Factor (connective + tissue_growth_factor)
Selected AbstractsConnective Tissue Growth Factor Promotes Fibrosis Downstream of TGF, and IL-6 in Chronic Cardiac Allograft RejectionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 2 2010A. J. Booth Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGF,, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGF, transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGF, expression remained unchanged, indicating that IL-6 effects may potentiate TGF,-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGF, and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR. [source] Connective tissue growth factor and cardiac fibrosisACTA PHYSIOLOGICA, Issue 3 2009A. Daniels Abstract Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart. [source] Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1, and ,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010D. Nowinski Abstract Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-, and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-,-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 ,-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1, and ,. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1, or , in presence or absence of TGF-,1. IL-1 suppressed basal and TGF-,-induced CTGF mRNA and protein expression. IL-1, and , inhibited TGF-,-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1, and , inhibited TGF-,-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-, activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-,-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226,1233, 2010. Published 2010 Wiley-Liss, Inc. [source] Src is a major signaling component for CTGF induction by TGF-,1 in osteoblasts,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010X. Zhang Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor ,1 (TGF-,1) where it acts as a downstream mediator of TGF-,1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-,1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-,1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-,1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-,1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-,1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-,1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. J. Cell. Physiol. 224: 691,701, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2009H. Takeuchi Background and Objective:, Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. Material and Methods:, Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription,polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. Results:, In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. Conclusion:, The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue. [source] The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1NEPHROLOGY, Issue 3 2007WEIER QI Summary: Aims: Smad7 is an inhibitory Smad that regulates transforming growth factor-, (TGF-,) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-,1. SMAD binding sites have been identified in both TGF-, and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-,1 in renal tubular cells is unknown. Methods: Human model of proximal tubular cells (HK-2 cells) was used and confirmed using a diabetic rat model. RT-PCR was performed to measure Smad7, TGF-,1 and Smad2 and ELISA was performed to measure active TGF-,1. CTGF or TGF-,1 was silenced in HK-2 cells using siRNA methodology. Results: TGF-,1 induced Smad7 in a time-dependent manner, peaking at 30 min (P < 0.0005) but sustained up to 24 hrs (p < 0.005). Conversely, CTGF reduced Smad7, which was maximal at 24 hrs (p < 0.05). This was supported by our in vivo data demonstrating that CTGF protein significantly increased while Smad7 mRNA level was reduced in a diabetic rat model. The basal expression level of Smad7 decreased in TGF-,1 silenced cells compared to cells transfected with non-specific siRNA (p < 0.0005). The basal expression level of Smad7 increased in CTGF silenced cells (p < 0.05), which was increased by TGF-,1 (p < 0.005). Both mRNA and protein levels of TGF-,1 decreased in CTGF silenced cells (p < 0.05 and p < 0.005 respectively) accompanied by reduction in Smad2 mRNA level in CTGF silenced cells. Conclusions: Smad7 is induced rapidly by TGF-,1 limiting the response to TGF-,1. CTGF likely plays a key role in promoting TGF-,1 activity by decreasing the availability of Smad7 and increasing Smad2. [source] Expression of connective tissue growth factor is in agreement with the expression of VEGF, VEGF-C, -D and associated with shorter survival in gastric cancerPATHOLOGY INTERNATIONAL, Issue 11 2007Luying Liu Connective tissue growth factor (CTGF) is believed to be a multifunctional signaling modulator involved in a wide variety of biological or pathological processes including carcinogenesis. The role of CTGF in gastric cancer (GC) has not been reported so far. In the present study the expression of CTGF, vascular endothelial growth factor (VEGF), VEGF-C and VEGF-D on immunohistochemistry in GC and the correlation between the expression of CTGF and VEGF, VEGF-C, VEGF-D were examined, along with the correlation between the expression of CTGF and clinicopathological parameters, as well as survival of the patients with GC. The expression of CTGF was significantly in agreement with expression of VEGF, VEGF-C and VEGF-D (, and P, respectively: 0.538, P < 0.001; 0.502, P < 0.001; 0.558, P < 0.001). High CTGF expression was significantly associated with lymph nodes metastasis (P = 0.038) and lower postoperative 5 year overall survival rates (23.9%) compared with those patients with low CTGF expression (48.4%, P = 0.0035). The present findings suggest that CTGF is a useful prognostic marker for GC. High CTGF expression is associated with the risk of lymph nodes metastasis and a poor survival time in GC. [source] Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosisARTHRITIS & RHEUMATISM, Issue 5 2010Sonali Sonnylal Objective Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor , (TGF,) signaling or through distinct signal transduction pathways. Methods We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen ,2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. Results Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGF,. Conclusion These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis. [source] Dabigatran, a direct thrombin inhibitor, demonstrates antifibrotic effects on lung fibroblastsARTHRITIS & RHEUMATISM, Issue 11 2009Galina S. Bogatkevich Objective Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor 1 (PAR-1) and induces a myofibroblast phenotype in normal lung fibroblasts resembling the phenotype of scleroderma lung myofibroblasts. We undertook this study to investigate whether a selective direct thrombin inhibitor, dabigatran, interferes with signal transduction in human lung fibroblasts induced by thrombin and mediated via PAR-1. Methods Lung fibroblast proliferation was analyzed using the Quick Cell Proliferation Assay. Expression and organization of ,-smooth muscle actin (,-SMA) was studied by immunofluorescence staining and immunoblotting. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Connective tissue growth factor (CTGF) and type I collagen expression was analyzed on Western blots. Results Dabigatran, at concentrations of 50,1,000 ng/ml, inhibited thrombin-induced cell proliferation, ,-SMA expression and organization, and the production of collagen and CTGF in normal lung fibroblasts. Moreover, when treated with dabigatran (1 ,g/ml), scleroderma lung myofibroblasts produced 6-fold less ,-SMA, 3-fold less CTGF, and 2-fold less type I collagen compared with untreated cells. Conclusion Dabigatran restrains important profibrotic events in lung fibroblasts and warrants study as a potential antifibrotic drug for the treatment of fibrosing lung diseases such as scleroderma lung disease and idiopathic pulmonary fibrosis. [source] Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagenARTHRITIS & RHEUMATISM, Issue 7 2009Markella Ponticos Objective Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor , (TGF,) and is a mediator of some profibrotic effects of TGF, in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I ,2) in this mouse model and in human pulmonary fibroblasts. Methods Transgenic mice that were carrying luciferase and ,-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts. Results In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by ,25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling. Conclusion Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc. [source] Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damageARTHRITIS & RHEUMATISM, Issue 5 2006E. N. Blaney Davidson Objective Characteristics of osteoarthritis (OA) include cartilage damage, fibrosis, and osteophyte formation. Connective tissue growth factor (CTGF; also known as CCN2), is found in high levels in OA chondrocytes and is frequently involved in fibrosis, bone formation, and cartilage repair. The present study was therefore undertaken to investigate the potential role of CTGF in OA pathophysiology. Methods We transfected the synovial lining of mouse knee joints with a recombinant adenovirus expressing human CTGF and measured synovial fibrosis and proteoglycan content in cartilage on days 1, 3, 7, 14, and 28. Messenger RNA (mRNA) expression in synovium and cartilage was measured on days 3, 7, and 21. Results CTGF induced synovial fibrosis, as indicated by accumulation of extracellular matrix and an increase in procollagen type I,positive cells. The fibrosis reached a maximum on day 7 and had reversed by day 28. Levels of mRNA for matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), and transforming growth factor , were elevated in the fibrotic tissue. TIMP-1 expression was elevated on day 3, while expression of other genes did not increase until day 7 or later. CTGF induced proteoglycan depletion in cartilage as early as day 1. Maximal depletion was observed on days 3,7. Cartilage damage was reduced by day 28. A high level of MMP-3 mRNA expression was found in cartilage. CTGF overexpression did not induce osteophyte formation. Conclusion CTGF induces transient fibrosis that is reversible within 28 days. Overexpression of CTGF in knee joints results in reversible cartilage damage, induced either by the high CTGF levels or via factors produced by the CTGF-induced fibrotic tissue. [source] Expression and regulation of connective tissue growth factor by transforming growth factor , and tumour necrosis factor , in fibroblasts isolated from strictures in patients with Crohn's diseaseBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 10 2006D. Beddy Background: Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and extracellular matrix production. Fibroblasts may initiate stricture formation in Crohn's disease through overexpression of CTGF. Stricturing that occurs in patients with Crohn's disease after treatment with anti-tumour necrosis factor (TNF) , may be due to dysregulation of CTGF homeostasis. The aim of this study was to examine CTGF expression and regulation in fibroblasts isolated from patients with Crohn's disease. Methods: Fibroblasts were isolated by a primary explant technique from serosal biopsies of strictured segments of bowel in eight patients undergoing resection for Crohn's disease and from normal colon in seven patients having resection for benign or malignant colorectal disease. Cells were stimulated with transforming growth factor (TGF) , and TNF-,. CTGF protein and mRNA expression were measured by western blotting and real-time polymerase chain reaction respectively. Results: Mean(s.d.) CTGF protein expression in strictured Crohn's fibroblasts was higher than that in normal fibroblasts (56·5(9·7) versus 17·0(10·0) respectively; P = 0·011). In normal and strictured Crohn's fibroblasts, culture with TGF-, increased CTGF protein and mRNA expression. Co-culture of normal fibroblasts with TNF-, suppressed TGF-,-stimulated CTGF expression. Conclusion: Increased expression of CTGF in strictured Crohn's fibroblasts underlies its role in fibrosis. TNF-, suppresses fibrosis by downregulating fibroblast CTGF expression, an effect that may be lost following anti-TNF-, treatment, thereby promoting stricture formation. Copyright © 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] The role of CTGF in the diabetic rat retina and its relationship with VEGF and TGF-,2, elucidated by treatment with CTGFsiRNAACTA OPHTHALMOLOGICA, Issue 6 2010Hongwei Yang Acta Ophthalmol. 2010: 88: 652,659 Abstract. Purpose:, The critical association of connective tissue growth factor (CTGF) with diabetic retinopathy (DR) remains to be clarified. We detected alterations in the gene and protein expression of CTGF and related cytokines, including vascular endothelial growth factor (VEGF) and transforming growth factor-,2 (TGF-,2), and their response to small interfering RNA (siRNA) targeting the CTGF (CTGFsiRNA) in the retina of diabetic rats. The relationships between CTGF, VEGF and TGF-,2 levels, as well as the degree of apoptosis in the diabetic retina, were also investigated. Methods:, Diabetes was induced in rats by the ,-cell toxin streptozotocin (STZ). Retinas were obtained from control and diabetic rats and similar animals treated with CTGFsiRNA by intravitreal injection. mRNA level and protein expression of CTGF, VEGF and TGF-,2 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and located by immunohistochemistry. Retinal apoptosis was detected by TUNEL staining. Results:, The levels of CTGF, VEGF and TGF-,2 and the number of TUNEL-positive nuclei were significantly higher in diabetic retinas than in control retinas (p < 0.01). The level of CTGF rose at 8 weeks, earlier than levels of VEGF and TGF-,2, which rose at 12 weeks after the onset of diabetes. The difference was significant (p < 0.05). siRNA-mediated inhibition of CTGF mRNA inhibited retinal VEGF and TGF-,2 and also resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and VEGF (p = 0.009), CTGF and TGF-,2 (p = 0.01), and apoptosis and these three cytokines (p < 0.01) in the rat retina early in diabetes. Conclusions:, These results suggest that the diabetes-mediated increase in CTGF upregulates VEGF and TGF-,2 expression and induces apoptosis in the retina. This elevation may be inhibited by treatment with CTGFsiRNA. Connective tissue growth factor may serve as a potential target for the prevention and treatment of DR. [source] Detection of connective tissue growth factor (CTGF) in human tear fluid: preliminary resultsACTA OPHTHALMOLOGICA, Issue 1 2003G. B. Van Setten Abstract. Purpose:, Connective tissue growth factor (CTGF) is one of the main regulators of fibrosis. We aimed to evaluate its presence in the human tear fluid of healthy individuals. Methods:, A total of 70 tear fluid samples were collected from eight volunteers prior to and after stimulation of reflex tears with onion vapour. Specific ELISA analysis was performed with goat IgG against human CTGF. Results:, Connective tissue growth factor was detected in seven samples (10%), with maximum levels of 17 ng/mL in basal tears. Induction of reflex tearing resulted in a fast and significant decrease of CTGF concentrations (r = , 0.95). No CTGF was detected in 90% of the samples. Conclusion:, Connective tissue growth factor may occur in tear fluid in healthy human eyes. This indicates a possible role for tear fluid CTGF in ocular surface fibrosis and wound healing. [source] Connective tissue growth factor and cardiac fibrosisACTA PHYSIOLOGICA, Issue 3 2009A. Daniels Abstract Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart. [source] Signalling and regulation of collagen I synthesis by ET-1 and TGF-,1FEBS JOURNAL, Issue 24 2005Angelika Horstmeyer Endothelin-1 (ET-1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET-1 signals are transduced by two receptor subtypes, the ETA- and ETB-receptors which activate different G, proteins. Here, we investigated the expression of both ET-receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA-receptor is the major ET-receptor subtype expressed. To determine further signalling intermediates, we inhibited G,i and three phospholipases. Pharmacologic inhibition of G,i, phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD), but not of phospholipase C,, abolished the increase in collagen I by ET-1. Inhibition of all phospholipases revealed similar effects on TGF-,1 induced collagen I synthesis, demonstrating involvement of PC-PLC and PLD in the signalling pathways elicited by ET-1 and TGF-,1. ET-1 and TGF-,1 each stimulated collagen I production and in an additive manner. ET-1 further induced connective tissue growth factor (CTGF), as did TGF-,1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF-,1 treatment, ET-1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET-1 treatment. Coincidentally at 5 days of permanent ET-1 stimulation, a switch in ET-receptor subtype expression to the ETB-receptor was observed. We conclude that the signalling pathways induced by ET-1 and TGF-,1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long-time stimulation by ET-1 resulted in a changed ET-receptor subtype ratio and in a biphasic CTGF induction. [source] Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotypeINTERNATIONAL JOURNAL OF CANCER, Issue 9 2009Katharine L. Sodek Abstract Ovarian cancer cells are present in malignant ascites both as individual cells and as multicellular spheroid aggregates. Although spheroid formation affords protection of cancer cells against some chemotherapeutic agents, it has not been established whether a relationship exists between invasive behavior and predisposition to spheroid formation. Aspects of spheroid formation, including cell-matrix adhesion, remodeling and contractility are characteristic myofibroblast-like behaviors associated with fibrosis that contribute to tumor growth and dissemination. We explored the possibility that cell behaviors that promote spheroid formation also facilitate invasion. Our analysis of 6 human ovarian cancer cell lines indicated that ovarian cancer cells possessing myofibroblast-like properties formed compact spheroids and invaded 3D matrices. These cells readily contracted collagen I gels, possessed a spindle-like morphology, and had elevated expression of genes associated with the TGF,-mediated fibrotic response and/or ,1 integrin function, including fibronectin (FN), connective tissue growth factor (CTGF/CCN2), lysyl oxidase (LOX1), tissue transglutaminase 2 (TGM2) and urinary plasminogen activator receptor (uPAR). Whereas cell aggregation was induced by TGF,, and by ,1-integrin overexpression and activation, these treatments did not stimulate the contractile activity required for spheroid compaction. The positive relationship found between compact spheroid formation and invasive behavior implies a preferential survival of an invasive subpopulation of ovarian cancer cells, as cells in spheroids are more resistant to several chemotherapeutics. Preventing the formation of ovarian cancer spheroids may represent a novel strategy to improve the efficacy of existing therapeutics. © 2008 Wiley-Liss, Inc. [source] CCN2, connective tissue growth factor, stimulates collagen deposition by gingival fibroblasts via module 3 and ,6- and ,1 integrinsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Edwin C.K. Heng Abstract CCN2, (connective tissue growth factor, CTGF) is a matricellular factor associated with fibrosis that plays an important role in the production and maintenance of fibrotic lesions. Increased collagen deposition and accumulation is a common feature of fibrotic tissues. The mechanisms by which CCN2/CTGF contributes to fibrosis are not well understood. Previous studies suggest that CTGF exerts some of its biological effects at least in part by integrin binding, though this mechanism has not been previously shown to contribute to fibrosis. Utilizing full length CCN2/CTGF, CCN2/CTGF fragments, and integrin neutralizing antibodies, we provide evidence that the effects of CCN2/CTGF to stimulate extracellular matrix deposition by gingival fibroblasts are mediated by the C-terminal half of CCN2/CTGF, and by ,6 and ,1 integrins. In addition, a synthetic peptide corresponding to a region of CCN2/CTGF domain 3 that binds ,6,1 inhibits the collagen-deposition assay. These studies employed a new and relatively rapid assay for CCN2/CTGF-stimulated collagen deposition based on Sirius Red staining of cell layers. Data obtained support a pathway in which CCN2/CTGF could bind to ,6,1 integrin and stimulate collagen deposition. These findings provide new experimental methodologies applicable to uncovering the mechanism and signal transduction pathways of CCN2/CTGF-mediated collagen deposition, and may provide insights into potential therapeutic strategies to treat gingival fibrosis and other fibrotic conditions. J. Cell. Biochem. 98: 409,420, 2006. © 2006 Wiley-Liss, Inc. [source] Role of TNF alpha and PLF in bone remodeling in a rat model of repetitive reaching and grasping,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Shobha Rani We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12,reaches/min, 60% maximum grip force, 2,h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNF, antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNF, treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNF, treatment attenuated task-induced increases in inflammatory cytokines (TNF,, IL-1,, and MIP2 in serum; TNF, in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNF, treatment. CTGF levels were unaffected by task performance or anti-TNF, treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNF,, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNF,, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro. J. Cell. Physiol. 225: 152,167, 2010. © 2010 Wiley-Liss, Inc. [source] Downregulation of a rheumatoid arthritis-related antigen (RA-A47) by ra-a47 antisense oligonucleotides induces inflammatory factors in chondrocytesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2003Takako Hattori Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor , (TNF,). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNF,. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNF,, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S -oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNF,-independent RA-A47 downregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNF, upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNF, synthesis. These observations indicate that downregulation of RA-A47 induces TNF,-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells. J. Cell. Physiol. 197: 94,102, 2003© 2003 Wiley-Liss, Inc. [source] Flutamide inhibits nifedipine- and interleukin-1,-induced collagen overproduction in gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2010H.-K. Lu Lu H-K, Tseng C-C, Lee Y-H, Li C-L, Wang L-F. Flutamide inhibits nifedipine- and interleukin-1,-induced collagen overproduction in gingival fibroblasts. J Periodont Res 2010; 45: 451,457. © 2010 John Wiley & Sons A/S Background and Objective:, To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin-1,- and nifedipine-induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Material and Methods:, Gingival fibroblasts from healthy subjects and patients with dihydropyridine-induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin-1, or both. The mRNA expression was examined using real-time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Results:, Interleukin-1, was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen ,1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen ,1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin-1,. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. Conclusion:, The data suggest that both nifedipine and interleukin-1, play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine-induced overgrowth cells. [source] Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2009H. Takeuchi Background and Objective:, Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. Material and Methods:, Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription,polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. Results:, In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. Conclusion:, The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue. [source] NeoHepatocytes From Alcoholics and Controls Express Hepatocyte Markers and Display Reduced Fibrogenic TGF-,/Smad3 Signaling: Advantage for Cell Transplantation?ALCOHOLISM, Issue 4 2010Sabrina Ehnert Background:, Liver transplantation is the only definitive treatment for end stage liver disease. Donor organ scarcity raises a growing interest in new therapeutic options. Recently, we have shown that injection of monocyte-derived NeoHepatocytes can increase survival in rats with extended liver resection. In order to apply this technology in humans with chronic liver diseases in an autologous setting, we generated NeoHepatocytes from patients with alcoholic liver disease and healthy controls and compared those to human hepatocytes. Methods:, We generated NeoHepatocytes from alcoholics with Child A and B cirrhosis and healthy controls. Hepatocytes marker expression and transforming growth factor (TGF)-, signaling was investigated by RT-PCR, Western blot, immunofluorescent staining, and adenoviral reporter assays. Glucose and urea was measured photometrically. Phase I and II enzyme activities were measured using fluorogenic substrates. Neutral lipids were visualized by Oil Red O staining. Results:, There was no significant difference in generation and yield of NeoHepatocytes from alcoholics and controls. Hepatocyte markers, e.g., cytokeratin18 and alcohol dehydrogenase 1, increased significantly throughout differentiation. Glucose and urea production did not differ between alcoholics and controls and was comparable to human hepatocytes. During differentiation, phase I and II enzyme activities increased, however remained significantly lower than in human hepatocytes. Fat accumulation was induced by treatment with insulin, TGF-, and ethanol only in differentiated cells and hepatocytes. TGF-, signaling, via Smad transcription factors, critically required for progression of chronic liver disease, was comparable among the investigated cell types, merely expression of Smad1 and -3 was reduced (,30 and ,60%) in monocytes, programmable cells of monocytic origin, and NeoHepatocytes. Subsequently, expression of TGF-, regulated pro-fibrogenic genes, e.g., connective tissue growth factor and fibronectin was reduced. Conclusions:, Generation of NeoHepatocytes from alcoholics, displaying several features of human hepatocytes, offers new perspectives for cell therapeutic approaches, as cells can be obtained repeatedly in a noninvasive manner. Furthermore, the autologous setting reduces the need for immunosuppressants, which may support recovery of patients which are declined for liver transplantation. [source] Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosisJOURNAL OF VIRAL HEPATITIS, Issue 9 2009E. Kovalenko Summary., Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-,) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-, bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection. [source] The differential regulation of Smad7 in kidney tubule cells by connective tissue growth factor and transforming growth factor-beta1NEPHROLOGY, Issue 3 2007WEIER QI Summary: Aims: Smad7 is an inhibitory Smad that regulates transforming growth factor-, (TGF-,) signaling. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of the fibrogenic effects of TGF-,1. SMAD binding sites have been identified in both TGF-, and CTGF promoters. The effect of CTGF on Smad7 expression and its role in the regulation of Smad7 induced by TGF-,1 in renal tubular cells is unknown. Methods: Human model of proximal tubular cells (HK-2 cells) was used and confirmed using a diabetic rat model. RT-PCR was performed to measure Smad7, TGF-,1 and Smad2 and ELISA was performed to measure active TGF-,1. CTGF or TGF-,1 was silenced in HK-2 cells using siRNA methodology. Results: TGF-,1 induced Smad7 in a time-dependent manner, peaking at 30 min (P < 0.0005) but sustained up to 24 hrs (p < 0.005). Conversely, CTGF reduced Smad7, which was maximal at 24 hrs (p < 0.05). This was supported by our in vivo data demonstrating that CTGF protein significantly increased while Smad7 mRNA level was reduced in a diabetic rat model. The basal expression level of Smad7 decreased in TGF-,1 silenced cells compared to cells transfected with non-specific siRNA (p < 0.0005). The basal expression level of Smad7 increased in CTGF silenced cells (p < 0.05), which was increased by TGF-,1 (p < 0.005). Both mRNA and protein levels of TGF-,1 decreased in CTGF silenced cells (p < 0.05 and p < 0.005 respectively) accompanied by reduction in Smad2 mRNA level in CTGF silenced cells. Conclusions: Smad7 is induced rapidly by TGF-,1 limiting the response to TGF-,1. CTGF likely plays a key role in promoting TGF-,1 activity by decreasing the availability of Smad7 and increasing Smad2. [source] Expression of connective tissue growth factor is in agreement with the expression of VEGF, VEGF-C, -D and associated with shorter survival in gastric cancerPATHOLOGY INTERNATIONAL, Issue 11 2007Luying Liu Connective tissue growth factor (CTGF) is believed to be a multifunctional signaling modulator involved in a wide variety of biological or pathological processes including carcinogenesis. The role of CTGF in gastric cancer (GC) has not been reported so far. In the present study the expression of CTGF, vascular endothelial growth factor (VEGF), VEGF-C and VEGF-D on immunohistochemistry in GC and the correlation between the expression of CTGF and VEGF, VEGF-C, VEGF-D were examined, along with the correlation between the expression of CTGF and clinicopathological parameters, as well as survival of the patients with GC. The expression of CTGF was significantly in agreement with expression of VEGF, VEGF-C and VEGF-D (, and P, respectively: 0.538, P < 0.001; 0.502, P < 0.001; 0.558, P < 0.001). High CTGF expression was significantly associated with lymph nodes metastasis (P = 0.038) and lower postoperative 5 year overall survival rates (23.9%) compared with those patients with low CTGF expression (48.4%, P = 0.0035). The present findings suggest that CTGF is a useful prognostic marker for GC. High CTGF expression is associated with the risk of lymph nodes metastasis and a poor survival time in GC. [source] Allograft Fibrosis,Unmasking the Players at the DanceAMERICAN JOURNAL OF TRANSPLANTATION, Issue 2 2010R. B. Mannon New data suggest that the pleiotropic cytokine interleukin-6 may be a key contributor to allograft fibrosis through a complex interaction with connective tissue growth factor (CTGF), a downstream effector of TGF-beta. See article by Booth et al on page 220. [source] Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGF, and IL-6 in Chronic Cardiac Allograft RejectionAMERICAN JOURNAL OF TRANSPLANTATION, Issue 2 2010A. J. Booth Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGF,, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGF, transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGF, expression remained unchanged, indicating that IL-6 effects may potentiate TGF,-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGF, and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR. [source] Anti-thrombin Therapy During Warm Ischemia and Cold Preservation Prevents Chronic Kidney Graft Fibrosis in a DCD ModelAMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2010F. Favreau Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran® (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-, (TGF-,) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival. [source] Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signalingARTHRITIS & RHEUMATISM, Issue 7 2010Cassie M. Tran Objective To investigate transforming growth factor , (TGF,) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Methods Real-time reverse transcription,polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGF,-mediated CTGF promoter activity. Results CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGF, treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGF,-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGF, was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGF, expression in the degenerated state. Conclusion TGF,, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc. [source] | |||||||||||||