1490

CTGF

BACKGROUND: Increasing evidence suggests that connective tissue growth factor (CTGF) is involved in the epithelial-to-mesenchymal transition (EMT). The exact intracellular events that drive this process, however, are not fully understood. In this study, we investigated the role of integrin-linked kinase (ILK) in mediating CTGF-induced EMT. METHODS: The expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin upon the stimulation by recombinant human CTGF (rhCTGF) in cultured human tubular epithelial cell line (HK-2) was detected by real-time RT-PCR and Western blot. Subsequently, the role of ILK was determined by using ILK siRNA. RESULTS: rhCTGF increased the mRNA expression of alpha-SMA significantly in a dose- and time-dependent manner, while E-cadherin mRNA decreased in a dose- and time-dependent manner. alpha-SMA protein was up-regulated after stimulation by 5 ng/ml CTGF for 96 h, and increased further after stimulation by 50 ng/ml. An immunocytochemical study showed that alpha-SMA was initially detectable at 48 h, and increased further at 72 h, while there was almost no alpha-SMA immunostaining observed in the control group at the same time point. E-cadherin protein was also down-regulated in a dose-dependent manner. Transfection of HK-2 cells with ILK-siRNA significantly attenuated rhCTGF-induced alpha-SMA induction and E-cadherin repression. CONCLUSION: Our study suggested that ILK mediated the effect of EMT in proximal tubular epithelial cells stimulated by CTGF.CI - (c) 2007 S. Karger AG, Basel.

Connective tissue growth factor (CTGF=CCN2), one of six members of cysteine-rich, secreted, heparin-binding proteins with a modular structure, is recognized as an important player in fibrogenic pathways as deduced from findings in non-hepatic tissues and emerging results from liver fibrosis. Collectively, the data show strongly increased expression in fibrosing tissues and transforming growth factor (TGF-beta)-stimulated expression in hepatocytes, biliary epithelial cells and stellate cells. Functional activity as a mediator of fibre-fibre, fibre-matrix and matrix-matrix interactions, as an enhancer of profibrogenic TGF-beta and several secondary effects owing to TGF-beta enhancement, and as a down-modulator of the bioactivity of bone morphogenetic protein-7 has been proposed. By changing the activity ratio of TGF-beta to its antagonist bone-morphogenetic protein-7, CTGF is proposed as a fibrogenic master switch for epithelial-mesenchymal transition. Consequently, knockdown of CTGF considerably attenuates experimental liver fibrosis. The spill-over of CTGF from the liver into the blood stream proposes this protein as a non-invasive reporter of TGF-beta bioactivity in this organ. Indeed, CTGF-levels in sera correlate significantly with fibrogenic activity. The data suggest CTGF as a multifaceted regulatory protein in fibrosis, which offers important translational aspects for diagnosis and follow-up of hepatic fibrogenesis and as a target for therapeutic interventions. In addition, CTGF-promoter polymorphism might be of importance as a prognostic genetic marker to predict the progression of fibrosis.

BACKGROUND: The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN: CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored. RESULTS: NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC. CONCLUSION: Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC.

OBJECTIVE: To construct a connective tissue growth factor (CTGF) and red fluorescent protein (RFP)-coexpressed renal tubular epithelial cell that can be used to quantitatively evaluate the CTGF-induced epithelial-mesenchymal transition (EMT). METHODS: The CTGF and RFP coding sequences were linked with a "2A" peptide, cloned into an expressing vector, and the HK-C2AR cell clone, which could express CTGF and RFP, were selected from the HK-2 cells after the recombinant plasmid transfection. Then, the CTGF-induced EMT in this cell line was determined, to further determine the association of RFP fluorescence intensity with the CTGF expression or CTGF-induced EMT. CTGF-specific ribonucleic acid (RNA) interference was also used to reconfirm the association. RESULTS: The constructed HK-C2AR cells could stably express RFP and CTGF proportionally, and the CTGF expressed in the cell line could induce EMT of cells, whereas the RFP expressed in the cell could exhibit bright red fluorescence after excitation. After the silence in CTGF, the RFP expression was also decreased, and the CTGF-induced EMT was also inhibited. CONCLUSION: The CTGF and RFP-coexpressing renal tubular epithelial cell, HK-C2AR, could be used to quantitatively evaluate CTGF-specific small interfering RNAs on EMT by quantitatively detecting the RFP expression. Also, this cell line could be used to quantitatively determine the mechanism of CTGF-induced EMT in renal tubular epithelial cells.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Transforming growth factor (TGF)-beta2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-beta2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-beta2, CTGF and gremlin can individually induce the expression of alpha-smooth muscle actin (alpha-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-beta2-induced expression of alpha-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of alpha-SMA, Fn, COL-I in HLECs. In conclusion, TGF-beta2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-beta2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IkappaB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IkappaB signaling each prohibited the EMT and tumor progression.

BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (P<0.05). We found significantly more positively expressed CTGF protein in ESCC tissues was than in normal adjacent esophageal tissues (P<0.01). Dual luciferase reporter gene assay showed that miR-145 can specifically bind with the 3'UTR of CTGF and significantly inhibit the luciferase activity by 55% (P<0.01). Up-regulation of miR-145 or down-regulation of CTGF can suppress the proliferation, migration, invasion, and EMT process of ESCC cells. CONCLUSIONS MiR-145 was significantly down-regulated in ESCC tissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

BACKGROUND: Tubulointerstitial inflammation and fibrosis are pathologic hallmarks of end-stage renal disease (ESRD). Here we have used DNA microarray technology to monitor the transcriptomic responses to murine unilateral ureteral obstruction (UUO) with a view to identifying molecular modulators of tubulointerstitial fibrosis. METHODS: Using Affymetrix Mu74Av2 microarrays, gene expression 4 and 10 days postobstruction was investigated relative to control contralateral kidneys. Candidate profibrogenic genes were further investigated in epithelial cells undergoing epithelial to mesenchymal transition (EMT) in vitro. RESULTS: mRNA levels for 1091 gene/EST sequences, of a total of 12,488 displayed on the microarray, were altered twofold or greater by days 4 and 10 postobstruction compared to contralateral control kidneys. Genes were categorised into functional groups, including modulators of cytoskeletal and extracellular matrix metabolism, cell growth, signalling, and transcription/translational events. Among the potentially profibrogenic genes, whose mRNA levels were increased after UUO, were fibroblast-inducible secreted protein (fisp-12), the murine homologue of connective tissue growth factor (CTGF), collagen XVIIIalpha1, secreted protein acidic and rich in cysteine (SPARC), and src-suppressed C-kinase substrate (SSeCKS). A sustained increase in fisp-12 mRNA level was observed during EMT induced by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF). CONCLUSION: Altered gene expression in murine UUO has been demonstrated. Increased expression of fisp-12, SPARC, and SSeCKS has been shown in response to TGF-beta1 treatment and during EMT, suggesting that these genes may offer potential therapeutic targets against tubulointerstitial fibrosis.

epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

BACKGROUND: Overexpression of connective tissue growth factor (CTGF) in alveolar type II epithelial (AT II) cells disrupts alveolar structure, causes interstitial fibrosis, and upregulates integrin-linked kinase (ILK). Whether CTGF-ILK signaling induces epithelial to mesenchymal transition (EMT) in AT II cells is unknown. METHODS: Transgenic mice with targeted overexpression of CTGF in AT II cells were generated utilizing the surfactant protein C (SP-C) gene promoter and doxycycline-inducible system. AT II cells were isolated from 4-wk-old CTGF-overexpressing (CTGF+) mice and control littermates, and cultured on Matrigel. Cells were transfected with ILK siRNA, and cell morphology and expression of cell differentiation markers were analyzed. RESULTS: The AT II cells from the control lungs grew in clusters and formed alveolar-like cysts and expressed SP-C. In contrast, the cells from CTGF+ lungs were spread and failed to form alveolar-like cysts. These cells expressed higher levels of CTGF, alpha smooth muscle actin (alpha-SMA), fibronectin and vimentin, the mesenchymal markers, suggesting EMT-like changes. Transfection with ILK siRNA not only dramatically attenuated ILK expression, but also decreased alpha-SMA expression as well as reversed cell morphological changes in CTGF+ AT II cells. CONCLUSION: Overexpression of CTGF induces EMT in mouse primary AT II cells and this is mediated by ILK.

OBJECTIVE: It is known that connective tissue growth factor (CTGF) and beta-catenin are involved in DN; however, the underlying molecular mechanisms remain unknown. Here we hypothesized that podocytes undergo epithelial-mesenchymal transition (EMT) in high-glucose condition and CTGF mediates high-glucose induced EMT by activating beta-catenin in podocytes. METHODS: The differentiated podocytes were cultured and divided into three groups: the normal glucose group (5 mmol/L glucose), the high-glucose group (30 mmol/L glucose), and the osmotic control group (5 mmol/L glucose supplemented with 25 mmol/L mannitol). The morphology of cultured podocytes was observed under phase contrast microscopy. To study the relevant markers of EMT, as well as CTGF and beta-catenin, the mRNA and protein expressions were analyzed by real-time PCR and western blotting, respectively. In addition, the effects of inhibition CTGF by anti-CTGF antibody on high-glucose-induced EMT and beta-catenin expression in podocytes were studied. RESULTS: High glucose not only induced phenotypic transition of podocytes but also increased the expression of CTGF and beta-catenin. Under high-glucose condition, podocytes underwent EMT, which were demonstrated by downregulation of nephrin and upregulation of desmin. Moreover, high-glucose-induced EMT and beta-catenin overexpression in podocytes were attenuated by anti-CTGF antibody. CONCLUSION: CTGF and beta-catenin are involved in the EMT of podocytes in diabetes. CTGF mediates high-glucose induced EMT through activation of beta-catenin in podocytes. CTGF inhibition may protect podocytes from EMT in diabetes.

Despite substantial progress in medical care, the morbidity rate of diabetic nephropathy (DN) remains high in patients with diabetes. Evidence suggests that connective tissue growth factor (CTGF) induced podocyte injury may contribute to DN and CTGF inhibition could reduce albuminuria. However, to date the mechanisms involved in the effect of CTGF on podocyte injury have not been fully understood. The aim of this study is to investigate the effects of therapeutic CTGF antibody on glomerular beta-catenin expression and podocyte epithelial-mesenchymal transition (EMT) in diabetic mice. C57BL/6J mice were randomly divided into three groups as the following: the control, DN, and DN treated by CTGF antibody group. DN was induced by a single intraperitoneal injection of streptozotocin and then CTGF antibody was administrated three times per week for 8 weeks. Urinary albumin excretion, mesangial proliferation and matrix deposition, and beta-catenin expression in glomeruli at mRNA and protein level were all increased in DN mice compared to that in the control. Besides, the development of EMT in podocytes from diabetic mice, demonstrated by the downregulation of nephrin and upregulation of desmin in glomeruli, was detected. Furthermore, blocking CTGF by specific antibody reduced albuminuria, prevented the overexpression of CTGF, as well as beta-catenin, in glomeruli and subsequently ameliorated podocyte EMT in DN mice. In summary, this study suggested that CTGF antibody protected podocytes against injury in DN mice by reducing beta-catenin overexpression and preventing podocyte EMT, which might provide new insight into the mechanism of CTGF inhibition in the treatment of DN. J. Cell. Biochem. 118: 3706-3712, 2017. (c) 2017 Wiley Periodicals, Inc.CI - (c) 2017 Wiley Periodicals, Inc.