Tuberc Respir Dis.  2016 Jul;79(3):143-152. 10.4046/trd.2016.79.3.143.

Apolipoprotein A1 Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition of Alveolar Epithelial Cells

• Baek AR
• Lee JM
• Seo HJ
• Park JS
• Lee JH
• Park SW
• Jang AS
• Kim DJ
• Koh ES
• Uh ST
• Kim YH
• Park CS

• KMID: 2326682
• DOI: http://doi.org/10.4046/trd.2016.79.3.143

Abstract

BACKGROUND
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the accumulation of excessive fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be a possible source of fibroblasts/myofibroblasts in IPF lungs. We have previously reported that apolipoprotein A1 (ApoA1) has anti-fibrotic activity in experimental lung fibrosis. In this study, we determine whether ApoA1 modulates TGF-β1-induced EMT in experimental lung fibrosis and clarify its mechanism of action.
METHODS
The A549 alveolar epithelial cell line was treated with TGF-β1 with or without ApoA1. Morphological changes and expression of EMT-related markers, including E-cadherin, N-cadherin, and α-smooth muscle actin were evaluated. Expressions of Smad and non-Smad mediators and TGF-β1 receptor type 1 (TβRI) and type 2 (TβRII) were measured. The silica-induced lung fibrosis model was established using ApoA1 overexpressing transgenic mice.
RESULTS
TGF-β1-treated A549 cells were changed to the mesenchymal morphology with less E-cadherin and more N-cadherin expression. The addition of ApoA1 inhibited the TGF-β1-induced change of the EMT phenotype. ApoA1 inhibited the TGF-β1-induced increase in the phosphorylation of Smad2 and 3 as well as that of ERK and p38 mitogen-activated protein kinase mediators. In addition, ApoA1 reduced the TGF-β1-induced increase in TβRI and TβRII expression. In a mouse model of silica-induced lung fibrosis, ApoA1 overexpression reduced the silica-mediated effects, which were increased N-cadherin and decreased E-cadherin expression in the alveolar epithelium.
CONCLUSION
Our data demonstrate that ApoA1 inhibits TGF-β1-induced EMT in experimental lung fibrosis.

Keyword

Apolipoprotein A-1; Transforming Growth Factor Beta1; Epithelial-Mesenchymal Transition; Pulmonary Fibrosis

MeSH Terms

Actins
Animals
Apolipoprotein A-I*
Apolipoproteins*
Cadherins
Epithelial Cells*
Epithelial-Mesenchymal Transition
Epithelium
Extracellular Matrix
Fibroblasts
Fibrosis
Idiopathic Pulmonary Fibrosis
Lung
Lung Diseases
Mice
Mice, Transgenic
Myofibroblasts
Phenotype
Phosphorylation
Protein Kinases
Pulmonary Fibrosis
Transforming Growth Factor beta1
Transforming Growth Factors
Actins
Apolipoprotein A-I
Apolipoproteins
Cadherins
Protein Kinases
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Figure 1 ApoA1 inhibits TGF-β1–induced morphological change in A549 cells. A549 cells were incubated with TGF-β1 (5 ng/mL) with or without ApoA1 (100 ng/mL) for 48 hours. (A) Untreated A549 cells showed a polygonal shape and intact cell-cell adhesion. (B) TGF-β1–treated cells showed a decrease in cell-cell adhesion and a greater spindle shape. (C) Co-treatment of ApoA1-treated cells maintained a polygonal shape and showed an intact cell-cell adhesion similar to the untreated cells (A–C, ×400). ApoA1: apolipoprotein A1; TGF-β1: transforming growth factor β1.

• 

  Figure 2 ApoA1 inhibits TGF-β1–induced EMT in A549 cells. A549 cells were treated with TGF-β1 (5 ng/mL) with or without ApoA1 (100 ng/mL) for 48 hours. Expressions of E-cadherin (green) (A), N-cadherin (green) (B), and α-SMA (red) were analyzed by immunofluorescence staining. Treatment with TGF-β1 decreased the expression of the epithelial marker, E-cadherin, and increased the expression of mesenchymal markers such as N-cadherin and α-SMA. ApoA1 inhibited TGF-β1–induced changes in EMT markers similar to control levels (A and B, ×400). ApoA1: apolipoprotein A1; TGF-β1: transforming growth factor β1; EMT: epithelial-to-mesenchymal transition; α-SMA: α-smooth muscle actin; DIC: differential interference contrast.

• 

  Figure 3 Effects of ApoA1 on EMT-related markers in A549 cells. A549 cells were incubated with TGF-β1 (5 ng/mL) in the absence of serum with or without ApoA1 (100 ng/mL) for up to 48 hours. Stimulation of cells by TGF-β1 down-regulated the epithelial marker, E-cadherin, and up-regulated the mesenchymal markers such as N-cadherin and α-SMA in a time dependent manner. β-Actin was used as a loading control (A). Densitometric analysis of band intensities for E-cadherin (B), N-cadherin (C), and α-SMA band (D). Each bar represents mean±standard error of at least three independent experiments. *p<0.05 versus same time of the TGF-β1–treated group. ApoA1: apolipoprotein A1; EMT: epithelial-to-mesenchymal transition; TGF-β1: transforming growth factor β1; α-SMA: α-smooth muscle actin.

• 

  Figure 4 Overexpression of ApoA1 attenuates silica-induced EMT in the mouse lung. Immunofluorescence staining for E-cadherin (green) (A), N-cadherin (green) (B), and pro-SPC (red) in the mouse lung. Treatment with silica decreased the expression of the epithelial marker, E-cadherin, and increased the expression of the mesenchymal markers such as N-cadherin and α-SMA in the WT mouse lung. Overexpression of ApoA1 inhibited TGF-β1–induced changes in EMT markers similar to silica non-treated WT levels (first-fourth columns, ×1,000; fifth columns, ×400). ApoA1: apolipoprotein A1; EMT: epithelial-to-mesenchymal transition; SPC: surfactant protein C; α-SMA: α-smooth muscle actin; WT: wild type; TGF-β1: transforming growth factor β1; DIC: differential interference contrast; TG: transgenic.

• 

  Figure 5 ApoA1 down-regulates the TGF-β1–induced Smad dependent signaling pathway. A549 cells were incubated with TGF-β1 (5 ng/mL) with or without ApoA1 (100 ng/mL) for 48 hours. (A) Phosphorylation of Smad2 and Smad3 occurred after TGF-β1 stimulation and co-treatment with ApoA1 down-regulated the TGF-β1–induced Smad signaling pathway. β-Actin was used as a loading control. Densitometric analysis of band intensities for phosphorylated Smad2 (B) and Smad3 (C). Each bar represents mean±standard error of at least three independent experiments. *p<0.05 versus same time of the TGF-β1–treated group. ApoA1: apolipoprotein A1; TGF-β1: transforming growth factor β1.

• 

  Figure 6 ApoA1 down-regulates the TGF-β1–induced non-Smad signaling pathways. A549 cells were incubated with TGF-β1 (5 ng/mL) with or without ApoA1 (100 ng/mL) for 48 hours. (A) Phosphorylation of ERK1/2 and p38 MAPK occurred after TGF-β1 stimulation and co-treatment with ApoA1 down–regulated the TGF-β1–induced non-Smad signaling pathways. β-Actin was used as a loading control. Densitometric analysis of band intensities for phosphorylated ERK1/2 (B) and p38 MAPK (C). Each bar represents mean±standard error of at least three independent experiments. *p<0.05 versus same time of the TGF-β1–treated group. ApoA1: apolipoprotein A1; TGF-β1: transforming growth factor β1; MAPK: mitogen-activated protein kinase.

• 

  Figure 7 ApoA1 decreases the expression of TβRI and TβRII in A549 cells. A549 cells were incubated with TGF-β1 (5 ng/mL) with or without ApoA1 (100 ng/mL) for 48 hours. (A) TGF-β1 increased both TβRI and TβRII expression and co-treatment with ApoA1 down-regulated TGF-β receptors. β-Actin was used as a loading control. Densitometric analysis of band intensities for TβRI (B) and TβRII (C). Each bar represents mean±SE of at least three independent experiments. *p<0.05 versus same time of the TGF-β1–treated group. ApoA1: apolipoprotein A1; TGF-β1: transforming growth factor β1; TβRI: TGF-β1 receptor type 1; TβRII: TGF-β1 receptor type 2.

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