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TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT).
Respir Res. 2005 Jun 09; 6:56.RR

Abstract

BACKGROUND

Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-beta1-mediated EMT.

METHODS

A549 cells were examined for evidence of EMT after treatment with TGF-beta1. 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-beta1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-beta1-mediated EMT was investigated using siRNA.

RESULTS

The data showed that TGF-beta1, but not TNF-alpha or IL-1beta, 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-beta1-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.

CONCLUSION

Our study shows that TGF-beta1 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.

Links

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Authors+Show Affiliations

Kasai H
Teijin Biomedical Laboratory, Medical Research Council Technology, 1-3 Burtonhole Lane, London, NW7 1AD, UK. hidenori.kasai@tech.mrc.ac.uk
Allen JT
No affiliation info available
Mason RM
No affiliation info available
Kamimura T
No affiliation info available
Zhang Z
No affiliation info available

MeSH

CadherinsCell DifferentiationCell LineDose-Response Relationship, DrugEpithelial CellsFibronectinsHumansMesenchymal Stem CellsPulmonary AlveoliRespiratory MucosaTransforming Growth Factor betaTransforming Growth Factor beta1

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15946381

Citation

Kasai, Hidenori, et al. "TGF-beta1 Induces Human Alveolar Epithelial to Mesenchymal Cell Transition (EMT)." Respiratory Research, vol. 6, 2005, p. 56.
Kasai H, Allen JT, Mason RM, et al. TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res. 2005;6:56.
Kasai, H., Allen, J. T., Mason, R. M., Kamimura, T., & Zhang, Z. (2005). TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respiratory Research, 6, 56.
Kasai H, et al. TGF-beta1 Induces Human Alveolar Epithelial to Mesenchymal Cell Transition (EMT). Respir Res. 2005 Jun 9;6:56. PubMed PMID: 15946381.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). AU - Kasai,Hidenori, AU - Allen,Jeremy T, AU - Mason,Roger M, AU - Kamimura,Takashi, AU - Zhang,Zhi, Y1 - 2005/06/09/ PY - 2004/12/08/received PY - 2005/06/09/accepted PY - 2005/6/11/pubmed PY - 2006/3/18/medline PY - 2005/6/11/entrez SP - 56 EP - 56 JF - Respiratory research JO - Respir Res VL - 6 N2 - BACKGROUND: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-beta1-mediated EMT. METHODS: A549 cells were examined for evidence of EMT after treatment with TGF-beta1. 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-beta1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-beta1-mediated EMT was investigated using siRNA. RESULTS: The data showed that TGF-beta1, but not TNF-alpha or IL-1beta, 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-beta1-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. CONCLUSION: Our study shows that TGF-beta1 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. SN - 1465-993X UR - https://www.unboundmedicine.com/medline/citation/15946381/TGF_beta1_induces_human_alveolar_epithelial_to_mesenchymal_cell_transition__EMT__ DB - PRIME DP - Unbound Medicine ER -