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Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells  [PDF]
Atsushi Yasukawa, Koa Hosoki, Masaaki Toda, Yasushi Miyake, Yuki Matsushima, Takahiro Matsumoto, Daniel Boveda-Ruiz, Paloma Gil-Bernabe, Mizuho Nagao, Mayumi Sugimoto, Yukiko Hiraguchi, Reiko Tokuda, Masahiro Naito, Takehiro Takagi, Corina N. D'Alessandro-Gabazza, Shigeru Suga, Tetsu Kobayashi, Takao Fujisawa, Osamu Taguchi, Esteban C. Gabazza
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064281
Abstract: Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.
Dioscorea alata Attenuates Renal Interstitial Cellular Fibrosis by Regulating Smad- and Epithelial-Mesenchymal Transition Signaling Pathways  [PDF]
Shu-Fen Liu,Shan-Yu Chang,Tao-Chen Lee,Lea-Yea Chuang,Jinn-Yuh Guh,Chien-Ya Hung,Tsung-Jen Hung,Yu-Ju Hung,Po-Yi Chen,Pei-fang Hsieh,Yu-Lin Yang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0047482
Abstract: Renal interstitial fibrosis is characterized by increased extracellular matrix (ECM) synthesis. Epithelial-mesenchymal transition (EMT) in kidneys is driven by regulated expression of fibrogenic cytokines such as transforming growth factor-beta (TGF-β). Yam, or Dioscorea alata (DA) is an important herb in Chinese medicine widely used for the treatment of clinical diabetes mellitus. However, the fibrosis regulatory effect of DA is unclear. Thus, we examined TGF-β signaling mechanisms against EMT in rat fibroblast cells (NRK-49F). The characterization of DA water-extracts used various methods; after inducing cellular fibrosis in NRK-49F cells by treatment with β-hydroxybutyrate (β-HB) (10 mM), we used Western blotting to examine the protein expression in the TGF-β-related signal protein type I and type II TGF-β receptors, Smads2 and Smad3 (Smad2/3), pSmad2 and Smad3 (pSmad2/3), Smads4, Smads7, and EMT markers. These markers included E-cadherin, alpha-smooth muscle actin (α-SMA), and matrix metalloproteinase-2 (MMP-2). Bioactive TGF-β and fibronectin levels in the culture media were determined using ELISA. Expressions of fibronectin and Snail transcription factor, an EMT-regulatory transcription factor, were assessed by immunofluorescence staining. DA extract dose-dependently (50–200 μg/mL) suppressed β-HB-induced expression of fibronectin in NRK-49F cells concomitantly with the inhibition of Smad2/3, pSmad2/3, and Smad4. By contrast, Smad7 expression was significantly increased. DA extract caused a decrease in α-SMA (α-smooth muscle actin) and MMP-2 levels, and an increase in E-cadherin expression. We propose that DA extract might act as a novel fibrosis antagonist, which acts partly by down regulating the TGF-β/smad signaling pathway and modulating EMT expression.
TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids
Astrid M Doerner, Bruce L Zuraw
Respiratory Research , 2009, DOI: 10.1186/1465-9921-10-100
Abstract: BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests.Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ1 induced transition but IL-1β enhanced the transition.Our results indicate, that TGFβ1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.Asthma is a chronic inflammatory disease of the airway, affecting approximately 10% of the general population [1]. Persistent asthma is characterized by structural changes termed airway remodeling. This ongoing remodeling and reconstruction of the asthmatic lung includes subepithelial fibrosis, myofibroblast hyperplasia, myocyte hyperplasia and/or hypertrophy, thickening of the lamina reticularis, and increased smooth muscle mass [2]. The more rapid decline in lung function over time in asthmatics is considered to be at least partly caused by this remodeling process. While the impact of corticosteroid treatment on airway re
Inhibitory Role of the KEAP1-NRF2 Pathway in TGFβ1-Stimulated Renal Epithelial Transition to Fibroblastic Cells: A Modulatory Effect on SMAD Signaling  [PDF]
In-geun Ryoo, Hunjoo Ha, Mi-Kyoung Kwak
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0093265
Abstract: Transforming growth factor β1 (TGFβ1) is a potent stimulator of epithelial-to-mesenchymal transition (EMT) and has been associated with chronic kidney diseases by activating profibrotic gene expression. In this study, we investigated the role of the KEAP1-NRF2 pathway, which is a master regulator of the cellular antioxidant system, in TGFβ1-stimulated EMT gene changes using human renal tubular epithelial HK2. Treatment with TGFβ1 enhanced the levels of reactive oxygen species (ROS) and TGFβ1-stimulated EMT gene changes, including an increase in profibrotic fibronectin-1 and collagen 1A1, were diminished by the antioxidant N-acetylcysteine. In HK2, TGFβ1 suppressed NRF2 activity and thereby reduced the expression of GSH synthesizing enzyme through the elevation of ATF3 level. Therefore, the activation of NRF2 signaling with sulforaphane effectively attenuated the TGFβ1-stimulated increase in fibronectin-1 and collagen 1A1. Conversely, the TGFβ1-EMT gene changes were further enhanced by NRF2 knockdown compared to the control cells. The relationship of NRF2 signaling and TGFβ1-EMT changes was further confirmed in a stable KEAP1-knockdown HK2, which is a model of pure activation of NRF2. The TGFβ1-mediated increase of collagen 1A1 and fibronectin-1 in KEAP1 knockdown HK2 was suppressed. In particular, TGFβ1-SMAD signaling was modulated in KEAP1 knockdown HK2: the TGFβ1-stimulated SMAD2/3 phosphorylation and SMAD transcriptional activity were repressed. Additionally, the protein level of SMAD7, an inhibitor of SMAD signaling, was elevated and the level of SMURF1, an E3 ubiquitin ligase for SMAD7 protein, was diminished in KEAP1 knockdown HK2. Finally, the inhibition of SMAD7 expression in KEAP1 knockdown HK2 restored TGFβ1 response, indicating that SMURF1-SMAD7 may be a molecular signaling linking the NRF2-GSH pathway to TGFβ1-EMT changes. Collectively, these results indicate that the KEAP1-NRF2 antioxidant system can be an effective modulator of TGFβ1-stimulated renal epithelial transition to fibroblastic cells through the SMUR1-SMAD7 signaling, and further implies the beneficial role of NRF2 in chronic renal diseases.
TNF- Regulates Natriuretic Peptides and Aquaporins in Human Bronchial Epithelial Cells BEAS-2B  [PDF]
Letizia Mezzasoma,Lucio Cagini,Cinzia Antognelli,Francesco Puma,Eugenio Pacifico,Vincenzo Nicola Talesa
Mediators of Inflammation , 2013, DOI: 10.1155/2013/159349
Abstract: Postoperative-fluid retention is a severe complication frequently reported in patients undergoing major surgical procedures. The complex network of molecules involved in such a severe surgery-induced condition remains poorly understood. Inflammation has been proposed among the various causes of fluid retention. Since TNF-α is one of the main proinflammatory cytokine initially released after major surgery, it is reasonable to assume its involvement in fluid overload. Here, we showed that TNF-α selectively regulates key molecules involved in fluids balance, such as natriuretic peptides (NPs) and aquaporins, in human bronchial epithelial cells BEAS-2B. In particular, we found that TNF-α induced a decrease of arial natriuretic peptide, natriuretic peptide receptor-1, aquaporin-1 and aquaporin-5 and an increase of brain natriuretic peptide with a different involvement of nuclear factor-κB and mitogen-activated protein kinases signaling pathway activation. Moreover, the observed changes in NPs expression, demonstrate inflammation as an additional cause of brain natriuretic peptide elevation, adding an important piece of information in the novel area of study regarding NPs and inflammation. Finally, we suggest that inflammation is one of the mechanisms of Aquaporin-1 and aquaporin-5 expression regulation. Therefore, in this exploratory study, we speculate that TNF-α might be involved in postoperative-fluid retention related to major surgery. 1. Introduction Weight gain with edema formation is frequently reported in patients undergoing major surgical procedures, with an incidence as high as 40% [1]. Postoperative weight gain and fluid overload have been associated with poor survival [2] and complications [3, 4]. The causes of fluid retention are various, and not completely clear. One of them could be related to the systemic response induced by surgical stress and operative trauma, regulated by a complex network of endocrine, neuronal, and immunological mechanisms [5–7]. Such surgery-induced reaction leads to an early hyperinflammatory status that is essential for tissue repair and host defense [5]. Cytokines are thought to play a pivotal role in the pathogenesis of surgical trauma. They have local effects of mediating and maintaining the inflammatory response to tissue injury and also initiate some of the systemic changes which occur [8–10]. After major surgery, is one of the main proinflammatory cytokines initially released in the damaged tissue where it stimulates the production and release of more cytokines, responsible for inducing the systemic changes
The Complex Interplay between ERK1/2, TGFβ/Smad, and Jagged/Notch Signaling Pathways in the Regulation of Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium Cells  [PDF]
Xiaoyun Chen, Wei Xiao, Wencong Wang, Lixia Luo, Shaobi Ye, Yizhi Liu
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0096365
Abstract: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is a major pathologic change in the development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. ERK1/2 pathway has been reported to play a key role in the carcinogenesis, cancer metastasis, and multiple fibrotic diseases. We hypothesized that ERK1/2 signaling could cross-interact with transforming growth factor β2 (TGFβ2)/Smad and Notch signaling pathways in the regulation of EMT in RPE cells. Here, we demonstrated that ERK1/2 signaling was activated in TGFβ2-induced EMT in human RPE cells, while blockade of the canonical TGFβ2/Smad2/3 signaling with SB431542 could not inhibit TGFβ2-induced the activation of ERK1/2. Meanwhile, blockade of ERK1/2 signaling with a specific MEK/ERK1/2 inhibitor U0126 strongly prevented TGFβ2-induced the downregulation of P-cadherin, and the upregulation of α-SMA, collagen type IV, N-cadherin and fibronectin in RPE cells. In addition, we also identified that blockade of ERK1/2 signaling could inhibit not only the canonical TGFβ/Smad signaling, but also the Jagged/Notch pathway. Finally, we found that blockade of Notch pathway with a specific inhibitor DAPT could inhibit TGFβ2-induced the activation of ERK1/2 pathway conversely. Therefore, our study provides evidence that ERK1/2 signaling can cross-interact with the canonical TGFβ/Smad and the Jagged/Notch signaling pathways in RPE cells EMT. ERK1/2 inhibitor may have therapeutic value in the prevention and treatment of PVR and other fibrotic diseases.
MIR-99a and MIR-99b Modulate TGF-β Induced Epithelial to Mesenchymal Plasticity in Normal Murine Mammary Gland Cells  [PDF]
Gianluca Turcatel, Nicole Rubin, Ahmed El-Hashash, David Warburton
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031032
Abstract: Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs. Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated. In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.
TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT)
Hidenori Kasai, Jeremy T Allen, Roger M Mason, Takashi Kamimura, Zhi Zhang
Respiratory Research , 2005, DOI: 10.1186/1465-9921-6-56
Abstract: A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA.The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes.Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.Idiopathic pulmonary fibrosis (IPF), the most common pulmonary fibrotic disorder, is a progressive and lethal disease of unknown etiology whose
Uremic Toxins Induce Kidney Fibrosis by Activating Intrarenal Renin–Angiotensin–Aldosterone System Associated Epithelial-to-Mesenchymal Transition  [PDF]
Chiao-Yin Sun, Shih-Chung Chang, Mai-Szu Wu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034026
Abstract: Background Uremic toxins are considered to have a determinant pathological role in the progression of chronic kidney disease. The aim of this study was to define the putative pathological roles of the renal renin–angiotensin–aldosterone system (RAAS) and renal tubular epithelial-to-mesenchymal transition (EMT) in kidney fibrosis induced by (indoxyl sulfate) IS and (p-cresol sulfate) PCS. Methods Mouse proximal renal tubular cells (PKSV-PRs) treated with IS or PCS were used. Half-nephrectomized B-6 mice were treated with IS or PCS for 4 weeks. In the losartan treatment study, the study animal was administrated with IS+losartan or PCS+losartan for 4 weeks. Results IS and PCS significantly activated the intrarenal RAAS by increasing renin, angiotensinogen, and angiotensin 1 (AT1) receptor expression, and decreasing AT2 receptor expression in vitro and in vivo. IS and PCS significantly increased transforming growth factor-β1 (TGF-β1) expression and activated the TGF-β pathway by increasing Smad2/Smad2-P, Smad3/Smad3-P, and Smad4 expression. The expression of the EMT-associated transcription factor Snail was increased by IS and PCS treatment. IS and PCS induced the phenotype of EMT-like transition in renal tubules by increasing the expression of fibronectin and α-smooth muscle actin and decreasing the expression of E-cadherin. Losartan significantly attenuated the expression of TGF-β1 and Snail, and decreased kidney fibrosis induced by IS and PCS in vivo. Conclusion Activating the renal RAAS/TGF-β pathway has an important pathological role in chronic kidney injury caused by IS and PCS. IS and PCS may increase Snail expression and induce EMT-like transition.
Lithium Attenuates TGF- -Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients  [PDF]
Marta Michalik,Katarzyna Anna Wójcik,Bogdan Jakie?a,Katarzyna Szpak,Ma?gorzata Pierzchalska,Marek Sanak,Zbigniew Madeja,Jaros?aw Czy?
Journal of Allergy , 2012, DOI: 10.1155/2012/206109
Abstract: Bronchial asthma is a chronic disorder accompanied by phenotypic transitions of bronchial epithelial cells, smooth muscle cells, and fibroblasts. Human bronchial fibroblasts (HBFs) derived from patients with diagnosed asthma display predestination towards TGF-β-induced phenotypic switches. Since the interference between TGF-β and GSK-3β signaling contributes to pathophysiology of chronic lung diseases, we investigated the effect of lithium, a nonspecific GSK-3β inhibitor, on TGF-β1-induced fibroblast to myofibroblast transition (FMT) in HBF and found that the inhibition of GSK-3β attenuates TGF-β1-induced FMT in HBF populations derived from asthmatic but not healthy donors. Cytoplasmically sequestrated β-catenin, abundant in TGF-β1/LiCl-stimulated asthmatic HBFs, most likely interacts with and inhibits the nuclear accumulation and signal transduction of Smad proteins. These data indicate that the specific cellular context determines FMT-related responses of HBFs to factors interfering with the TGF-β signaling pathway. They may also provide a mechanistic explanation for epidemiological data revealing coincidental remission of asthmatic syndromes and their recurrence upon the discontinuation of lithium therapy in certain psychiatric diseases. 1. Introduction Bronchial asthma is a chronic disorder characterized by airway inflammation, remodeling of the airways, and their hyperresponsiveness to environmental stimuli. It is accompanied by thickening of the bronchial walls, epithelial damage, subepithelial fibrosis, and the increased deposition of extracellular matrix proteins. The bulk of these changes results from the phenotypic transitions of bronchial epithelial cells, smooth muscle cells, and fibroblasts [1, 2]. In general, phenotypic shifts, such as epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT), play a crucial role during various physiological and pathological processes [3–5]. In particular, erroneous function of myofibroblasts, representing the contractile subset of fibroblasts which have undergone FMT, was found to be crucial for the development of asthma [6, 7]. FMT, which is also involved in wound contraction and participates in the development of fibrotic changes in several tissues [8–10], can be induced by a range of proinflammatory cytokines, in particular by factors belonging to the transforming growth factor β (TGF-β) family [11, 12]. On the other hand, an increase in the local concentration of TGF-β was observed in bronchoalveolar lavage fluid sampled from asthmatic patients [13], and prolonged exposure
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