J Korean Med Sci.  2007 Oct;22(5):898-904. 10.3346/jkms.2007.22.5.898.

Transtorming Growth Factor beta1 Induces Epithelial-to-Mesenchymal Transition of A549 Cells

• Kim JH
• Jang YS
• Eom KS
• Hwang YI
• Kang HR
• Jang SH
• Kim CH
• Park YB
• Lee MG
• Hyun IG
• Jung KS
• Kim DG

• KMID: 1713300
• DOI: http://doi.org/10.3346/jkms.2007.22.5.898

Abstract

Idiopathic pulmonary fibrosis (IPF) comprises an aggregate of mesenchymal cells. However, the cellular origin of these mesenchymal phenotypes remains unclear. Transforming growth factor beta1 (TGF-beta1) has been known as the main cytokine involved in the pathogenesis of IPF. We examined whether the potent fibrogenic cytokine TGF-beta1 could induce the epithelial-to-mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and determined whether snail expression is associated with the phenotypic changes observed in the A549 cells. EMT was investigated with cells morphology changes under phase-contrast microscopy, western blotting, and indirect immunofluorescence stains. E-cadherin and transcription factor, snail, were also evaluated by measuring mRNA levels using reverse transcriptase-polymerase chain rection (RT-PCR) analysis. The data showed that TGF-beta1 induced A549 cells with epithelial cell characteristics to undergo EMT in a concentration-dependent manner. Following TGF-beta1 treatment, A549 cells induced EMT characterized by cells morphological changes, loss of epithelial markers Ecaherin and cytokeratin, increased stress fiber reorganization by F-actin, and cytokeratin replacement by vimentin. Although IL-1beta failed to induce A549 cells to undergo EMT, the combination of TGF-beta1 and IL-1beta showed synergy effects in cells morphology changes and the expression of mesenchymal markers. The snail expression study using RT-PCR analysis provided that loss of E-cadherin expression was associated with snail expression. Stimulation of A54 cells with TGF-beta1 plus IL-1beta revealed a higher level of snail expression. Our data showed that EMT of A549 cells might be closely associated with snail expression.

Keyword

Transforming Growth Factor-beta1; A549; EMT; Snail

MeSH Terms

Actins/metabolism
Cadherins/metabolism
Cell Differentiation
Cell Line, Tumor
Dose-Response Relationship, Drug
Epithelium/*metabolism
Fluorescent Antibody Technique, Indirect
*Gene Expression Regulation, Neoplastic
Humans
Keratins/metabolism
Mesoderm/*metabolism
Microscopy, Fluorescence
Reverse Transcriptase Polymerase Chain Reaction
Transcription, Genetic
Transforming Growth Factor beta1/metabolism/*physiology
Vimentin/metabolism

Figure

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  Fig. 1 The concentration of TGF-β1 to induce A549 cells to undergo EMT. A549 cells were incubated with TGF-β1 at various concentrations for 48 hr. (A) Morphologic changes are observed with 0.05 ng/mL of TGF-β1, and TGF-β1 treated cells with concentration as high as 5 ng/mL show more fibroblast-like morphologic changes. a, control; b, 0.05 ng/mL; c, 0.5 ng/mL; d, 5 ng/mL TGF-β1. (B) Expression of epithelial marker E-cadherin and cytokeratin are down-regulated by TGF-β1 stimulation in a concentration-dependent manner.

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  Fig. 2 Epithelial-to-mesenchymal transition of A549 cells in vitro. A549 cells treated without (upper column) or with 5 ng/mL TGF-β1 (lower column) for 48 hr in serum-free medium. A549 cells changed to more elongated, fibroblast-like cells (A, F). Mesenchymal transition cells revealed loss of E-cadherin (B, G), cytokeratin (C, H), cytokeratin replacement by vimentin (D, I), and stress fiber reorganization by F-actin (E, F).

• 

  Fig. 3 The effects of IL-1β alone and both stimulation of TGF-β1 and IL-1β on A459 cells. A549 cells treated without (upper column) or with 2 ng/mL IL-1β (middle column) or both stimulation of 2 ng/mL IL-1β and 5 ng/mL TGF-β1 (lower column) for 48 hr in serum-free medium. IL-1β-alone stimulation displayed milder cells elongation and weaker E-cadherin expression than in control, but distinct morphologic changes such as TGF-β1 stimulation were not observed. Stimulation of both IL-1β and TGF-β1 showed loss of E-cadherin, cytokeratin replacement by vimentin and stress fiber reorganization by F-actin. E-cadherin (B, G, F), cytokeratin (C, H,M), vimentin (D, I, M), F-actin (E, J, O).

• 

  Fig. 4 The effects of TGF-β1 alone and stimulation of both TGF-β1 and IL-1β in A459 cells. A549 cells treated without (upper column) or with 0.5 ng/mL of TGFβ1 (middle column), or in combination of 2 ng/mL of IL-1β and 0.5 ng/mL of TGF-β1 (lower column) for 48 hr in serum-free medium. TGF-β1 stimulation revealed epithelial-to-mesenchymal transition. In combination of IL-1β plus TGF-β1, cells changed to more fibroblast-like morphology under phase contrast light microscopy and loss of E-cadherin expression, cytokeratin replacement by vimentin, and stress fiber reorganization by F-actin became more profound. E-cadherin (B, G, F), cytokeratin (C, H, M), vimentin (D, I, M), F-actin (E, J, O).

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  Fig. 5 Expression of snail transcription factor and the loss of E-cadherin expression. A549 cells were cultured for 48 hr without TGF-β1 (A), with 0.5 ng/mL TGF-β1 (B), and in a combination of 0.5 ng/mL TGF-β1 plus 2 ng/mL IL-1β (C). 0.5 ng/mL TGF-β1 began to reveal loss of E-cadherin expression and increasing snail expression, and addition with IL-1β shows the elevation of snail expression.

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