Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.     

Neuropilin-2 expression promotes TGF-β1-mediated epithelial to mesenchymal transition in colorectal cancer cells

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.     

Chidamide alleviates TGF-β-induced epithelial–mesenchymal transition in lung cancer cell lines

Transforming growth factor-β (TGF-β)-induced epithelial–mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.     

SKIP is required for TGF-β1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-β1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-β1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-β1-induced EMT, and the cells were sensitized to the TGF-β1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-β1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-β1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-β1 in transformed cells.     

PCBP1 is an important mediator of TGF-β-induced epithelial to mesenchymal transition in gall bladder cancer cell line GBC-SD

Gall bladder carcinoma (GBC) is the seventh most common cancer across the globe and the most common malignancy of the biliary tract. Most GBC related deaths occur due to secondary progression and metastasis to distant organs. Epithelial–mesenchymal transition (EMT) is an important pre-requisite for tumor metastasis, however its mechanism in GBC has not yet been defined. Using the GBC-SD cell line, we have uncovered an important mediator, poly r(C) binding protein-1 (PCBP1), of transforming growth factor-beta (TGF-β)-induced EMT in GBC. Our results show that TGF-β treatment resulted in PCBP1 phosphorylation in accordance with similar observation in other model systems. We further showed through gain- and loss-of-function assays that PCBP1 expression levels regulate the capacity of GBC-SD cells to migrate and invade in vitro. Finally, our results showed that PCBP1 expression levels also regulate generation of CD44⁺CD24^? progenitor cell population in GBC-SD cells after TGF-β treatment. Cumulatively, our results indicate, pending further validation, that PCBP1 might be a prognostic marker for GBC metastasis.     

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.     

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells

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 TGFβ2-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 TGFβ2-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 TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-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 TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-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.     

TGF-β1-induced epithelial–mesenchymal transition in lung cancer cells involves upregulation of miR-9 and downregulation of its target,E-cadherin

Background

TGF-β1 plays an important role in the epithelial–mesenchymal transition (EMT) of epithelial cancers, including non-small cell lung cancer (NSCLC). While the full underlying mechanism remains unclear, miR-9 is known to play a critical role in the regulation of NSCLC cell invasion. We tested whether miR-9 targets E-cadherin and thus affects TGF-β1-induced EMT in NSCLC cells by assessing the expression levels of miR-9 and E-cadherin for NSCLC patients and then verifying the targeting of E-cadherin by miR-9 using the dual luciferase reporter system.

Results

MiR-9 was significantly upregulated in NSCLC tissues compared with its level in adjacent normal tissues. The expression of E-cadherin in NSCLC tissues was significantly decreased. In addition, we found that TGF-β1 significantly upregulated the expression of miR-9 and downregulated the expression of E-cadherin. E-cadherin was confirmed as a direct target gene of miR-9. Using an miR-9 inhibitor reversed the TGF-β1-mediated inhibition of E-cadherin expression and upregulation of the mesenchymal marker α-SMA. TGF-β1 significantly induced cell invasion, and this effect was significantly inhibited by miR-9 inhibitors.

Conclusions

TGF-β1 induced EMT in NSCLC cells by upregulating miR-9 and downregulating miR-9’s target, E-cadherin.

     

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.     

IGF-1-induced epithelial–mesenchymal transition in MCF-7 cells is mediated by MUC1

Metastases are the major cause of death from cancer. IGF-1 signaling pathway has been shown to have strong implication in the epithelial–mesenchymal transition (EMT) process. However, the mechanisms of how IGF-1 promotes EMT have not been fully elucidated. Mucin 1 (MUC1), a transmembrane glycoprotein, engages in multiple cancer-related signaling pathways and functions as an oncoprotein that contributes to metastases. Here we provide evidence showing that IGF-1 upregulates MUC1 expression in MCF-7 cells in a PI3K/Akt signaling pathway-dependent manner. The overexpression of MUC1 is critical for IGF-1-induced EMT of MCF-7 cells because the knockdown of MUC1 prevented the EMT of MCF-7 cells as demonstrated by various EMT markers including the expression of E-cadherin, N-cadherin, vimentin, fibronectin and the nuclear translocalization of β-catenin. On the other hand, the knockdown of MUC1 had no impact on IGF-1-induced activation of PI3K/Akt or MAPK. In summary, our study demonstrated MUC1 as a critical downstream effector that mediates IGF-1-induced EMT of MCF-7 cells and suggested that MUC1 might be a potential therapeutic target for preventing tumor metastases.     

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Lefty antagonises TGF-β1 induced epithelial-mesenchymal transition in tubular epithelial cells

Lefty is a novel member of the transforming growth factor (TGF) supergene family which has the potential to antagonise actions of TGF-β1 - the main factor driving fibrotic disease in the kidney and in other organs. TGF-β1 can induce fibrosis through several mechanisms, including epithelial-mesenchymal transition (EMT) which contributes to myofibroblast accumulation in the renal interstitium. This study examined whether Lefty can antagonise TGF-β1 mediated EMT. A rat tubular epithelial cell line (NRK52E) was stably transfected with a Lefty expression plasmid (52E-Lefty) or control plasmid (52E-Control). 52E-Control cells underwent TGF-β1 induced EMT with up-regulation of α-smooth muscle actin (α-SMA), down-regulation of E-cadherin, and transition to an elongated fibroblast-like morphology. In contrast, 52E-Lefty cells were substantially protected from TGF-β1 induced EMT. Analysis of signalling pathways showed that 52E-Lefty cells had a marked reduction in TGF-β1 induced Smad activity and suppression of the secondary phase of JNK (but not p38) signalling. Treatment of NRK52E cells with a JNK inhibitor was shown to suppress TGF-β1 induced EMT. In conclusion, Lefty can antagonise TGF-β1 mediated EMT in renal tubular epithelial cells. Lefty may have potential as an anti-fibrotic molecule in the treatment of renal fibrosis.