7042

TGFB2

BACKGROUND: epithelial mesenchymal transition (EMT) of postoperative remnants of lens epithelial cells (LECs) can lead to posterior capsule opacification. This study was designed to determine the effect of signaling pathways that contribute to TGF-beta2-mediated EMT in human lens epithelial B-3 cells (HLEB-3 cells). METHODS: The HLEB-3 cells were cultured and stimulated with TGF-beta2 at different concentrations for an indicated time. The effect of TGF-beta2 on cell cycle distribution was measured by flow cytometry. Western blot and immunofluorescence were used to analyze changes in connexin 43, fibronectin, desmin and integrin beta(1) protein expression associated with EMT in HLEB-3 cells. Activation of phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was also detected by Western blot. RESULTS: The cell cycle progression of HLEB-3 cells was limited, and the cells underwent morphological alteration after treatment with TGF-beta2. Stimulation of HLEB-3 cells with TGF-beta(2) suppressed connexin 43 protein expression, increased fibronectin, desmin and integrin beta1 protein expression. TGF-beta2 activated PI3K/Akt in a time-dependent manner, but not extracellular signal-regulated kinase and p38 MAPK. The activation of PI3K/Akt was necessary for the TGF-beta(2)-stimulated downregulation of connexin 43, which in turn was necessary for TGF-beta2-induced EMT in HLEB-3 cells. CONCLUSIONS: TGF-beta(2) is a potent growth factor for LEC EMT. TGF-beta(2)-induced EMT in LECs is mediated by the downregulation of connexin 43, which is regulated through the PI3K/Akt pathway.CI - (c) 2008 S. Karger AG, BaselFAU - Yao, Ke

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.

The epithelial-mesenchymal transition (EMT) is a key process in tumor invasion and migration. Transforming growth factor-betas (TGF-betas) are multifunctional growth factors and powerful modulators of the EMT. Here, we investigated the relationship between TGF-beta expression and invasion by treated triple-negative breast cancer (TNBC) cells. Our results show that invasion capacity of TNBC cells was markedly higher than that of non-TNBC cells. In addition, EMT-related gene signatures, including vimentin (vim), fibronectin (FN), snail, and slug were highly expressed in TNBC cells. Interestingly, our results show that TGF-beta1 and beta2 mRNA expression levels were higher in TNBC cells than those in non-TNBC cells. Thus, we examined the effect of the TGF-beta receptor I/II inhibitor LY2109761 on EMT-related gene expression and cell motility. Our data show that vim, FN, and slug mRNA expression levels dose-dependently decreased in response to LY2109761. TNBC cell motility also decreased in response to LY2109761. Finally, we investigated the effect of LY2109761 on TGF-beta1 or TGF-beta2-induced E-cadherin (E-cad), vim, and FN mRNA and protein expression. The reduction in E-cad and induction of vim and FN expression by TGF-beta1 or TGF-beta2 were completely reversed by LY2109761 treatment in HCC1806 TNBC cells. Taken together, we demonstrated that elevated TGF-beta expression triggers invasion and migration by TNBCs through the EMT process. Inhibiting the TGF-beta signaling pathway is considered a promising therapeutic strategy for treating TNBC.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

The present study aimed to investigate whether the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is involved in the transforming growth factor beta2 (TGF-beta2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial (HLE) cells. HLEB-3 cells were cultured and stimulated with 10 ng/ml TGF-beta2 for 24 h. Western blotting was then performed to analyze the expression levels of connexin 43 and fibronectin, and the activities of Akt and mTOR. Confocal cell immunofluorescence was used to observe the expression of phosphorylated (p)-Akt. The toxicity of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) was assessed using a Cell Counting Kit-8 assay, and inhibition investigations were performed using a PI3K inhibitor. The expression of connexin 43 was suppressed and the expression of fibronectin was increased when the cells were stimulated with 10 ng/ml TGF-beta2 for 24 h. In addition, Akt and mTOR were activated during TGF-beta2-induced EMT. Treatment of with LY294002 (20 microM) inhibited the activation of Akt and mTOR and effectively prevented TGF-beta2-induced EMT in the HLECs. Therefore, the results of the present study indicated that TGF-beta2 induces EMT by activating the PI3K/Akt/mTOR signaling pathway in cultured HLECs.

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFbeta2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFbeta2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFbeta2. RAGE overexpression significantly enhanced the TGFbeta2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFbeta2-mediated EMT response. This was accompanied by a reduction in TGFbeta2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFbeta2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

The pathological development of lens epithelial cells (LECs) leads to posterior capsular opacification (PCO). This study was undertaken to investigate the effects of microRNA-486-5p (miR-486-5p) on TGF-beta2-induced proliferation, invasion and epithelial-mesenchymal transition (EMT) in the lens epithelial cell line SRA01/04, and to explore the underlying molecular mechanisms. The expression of miR-486-5p in TGF-beta2-induced SRA01/04 cells was down-regulated, and the expression of Smad2, p-Smad2 and p-Smad3 was up-regulated. A dual-luciferase reporter assay revealed that miR-486-5p directly targets the 30'-UTR of Smad2. MiR-486-5p mimic transfection markedly down-regulated the expression levels of Smad2, thus inhibiting the expression of p-Smad2 and p-Smad3. MiR-486-5p overexpression in SRA01/04 cells markedly suppressed TGF-beta2-induced proliferation and invasion, inhibited protein expression of CDK2 and CDK4, down-regulated fibronectin, alpha-SMA and vimentin and up-regulated E-cadherin; these effects were partly reversed by Smad2 overexpression. In short, these data show that miR-486-5p overexpression can inhibit TGF-beta2-induced proliferation, invasion and EMT in SRA01/04 cells by repressing Smad2/Smad3 signalling, implying that miR-486-5p may be an effective target to interfere in the progression of PCO.

Human lens epithelial cells (HLE) undergo mesenchymal transition and become fibrotic during posterior capsule opacification (PCO), which is a frequent complication after cataract surgery. TGF-beta2 has been implicated in this fibrosis. Previous studies have focused on the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in fibrotic diseases, but the role of HIF-1alpha in the TGF-beta2-mediated fibrosis in HLE is not known. TGF-beta2 treatment (10 ng/mL, 48 h) increased the HIF-1alpha levels along with the EMT markers in cultured human lens epithelial cells (FHL124 cells). The increase in HIF-1alpha corresponded to an increase in VEGF-A in the culture medium. However, exogenous addition of VEGF-A (up to 10 ng/mL) did not alter the EMT marker levels in HLE. Addition of a prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, up to 10 microM), enhanced the levels of HIF-1alpha, and secreted VEGF-A but did not alter the EMT marker levels. However, treatment of cells with a HIF-1alpha translational inhibitor, KC7F2, significantly reduced the TGF-beta2-mediated EMT response. This was accompanied by a reduction in the ERK phosphorylation and nuclear translocation of Snail and Slug. Together, these data suggest that HIF-1alpha is important for the TGF-beta2-mediated EMT of human lens epithelial cells.CI - (c) 2018 Wiley Periodicals, Inc.

Glucocorticoids (GCs) such as dexamethasone (DEX) are administered as cancer cotreatment for palliative purposes due to their proapoptotic effects in lymphoid cancer and limited side effects associated with cancer growth and chemotherapy. However, there is emerging evidence that GCs induce therapy resistance in most epithelial tumors. Our recent data reveal that DEX promotes the progression of pancreatic ductal adenocarcinoma (PDA). In the present study, we examined 1 primary and 2 established PDA cell lines, and 35 PDA tissues from patients who had received (n=14) or not received (n=21) GCs prior to surgery. Through microRNA microarray analysis, in silico, and RTqPCR analyses, we identified 268 microRNAs differentially expressed between DEXtreated and untreated cells. With a focus on cancer progression, we selected miR132 and its target gene, transforming growth factor-beta2 (TGFbeta2), as top candidates. miR132 mimics directly bound to the 3' untranslated region (3'UTR) of a TGFbeta2 luciferase construct and enhanced expression, as shown by increased luciferase activity. By contrast, DEX inhibited miR132 expression via promoter methylation. miR132 mimics also reduced DEXinduced clonogenicity, migration and expression of vimentin and Ecadherin in vitro and in tumor xenografts. In patients, GC intake prior to surgery enhanced global hypermethylation and expression of TGFbeta2 in tissues; expression of miR132 was detected but could not be quantified. Our results demonstrate that DEXmediated inhibition of miR132 is a key mediator in the progression of pancreatic cancer, and the findings provide a foundation for miRNAbased therapies.

epithelial-mesenchymal transition (EMT) plays important roles in various physiological and pathological processes, and is regulated by signaling pathways mediated by cytokines, including transforming growth factor beta (TGFbeta). Embryonic endothelial cells also undergo differentiation into mesenchymal cells during heart valve formation and aortic maturation. However, the molecular mechanisms that regulate such endothelial-mesenchymal transition (EndMT) remain to be elucidated. Here we show that TGFbeta plays important roles during mural differentiation of mouse embryonic stem cell-derived endothelial cells (MESECs). TGFbeta2 induced the differentiation of MESECs into mural cells, with a decrease in the expression of the endothelial marker claudin 5, and an increase in expression of the mural markers smooth muscle alpha-actin, SM22alpha and calponin, whereas a TGFbeta type I receptor kinase inhibitor inhibited EndMT. Among the transcription factors involved in EMT, Snail was induced by TGFbeta2 in MESECs. Tetracycline-regulated expression of Snail induced the differentiation of MESECs into mural cells, whereas knockdown of Snail expression abrogated TGFbeta2-induced mural differentiation of MESECs. These results indicate that Snail mediates the actions of endogenous TGFbeta signals that induce EndMT.

epithelial-mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum-free medium. Single cell-derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E-cadherin and beta-catenin from intercellular junctions, coupled with nuclear translocation of beta-catenin and LEF-1. Large-colony-forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of alpha-SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of alpha-SMA was promoted by high-density cultures and their conditioned media, which contained cell density-dependent levels of TGF-beta1, TGF-beta2, GM-CSF, and IL-1alpha. Exogenous TGF-beta1 induced alpha-SMA positive cells in a low-density culture, while TGF-beta1 neutralizing antibody partially inhibited alpha-SMA expression in a high-density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad-mediated TGF-beta-signaling.CI - Copyright (c) 2012 Wiley Periodicals, Inc.

epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.