Tuberc Respir Dis.  2011 Jun;70(6):462-473.

Inhibition of Plasminogen Activator Inhibitor-1 Expression in Smoke-Exposed Alveolar Type II Epithelial Cells Attenuates Epithelial-Mesenchymal Transition

Affiliations
  • 1Department of Internal Medicine, Yeouido St Mary's Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea. jssong@catholic.ac.kr

Abstract

BACKGROUND
Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the mechanism of the association remains obscure. There is evidence demonstrating that plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. This study was to determine whether the administration of small interfering RNA (siRNA) targeting PAI-1 or PAI-1 inhibitor to the cigarette smoking extract (CSE)-exposed rat alveolar type II epithelial cells (ATII cells) limits the epithelial-mesenchymal transition (EMT).
METHODS
ATII cells were isolated from lung of SD-rat using percoll gradient method and cultured with 5% CSE. The EMT was determined from the ATII cells by measuring the real-time RT PCR and western blotting after the PAI-1 siRNA transfection to the cells and after administration of tiplaxtinin, an inhibitor of PAI-1. The effect of PAI-1 inhibitor was also evaluated in the bleomycin-induced rats.
RESULTS
PAI-1 was overexpressed in the smoking exposed ATII cells and was directly associated with EMT. The EMT from the ATII cells was suppressed by PAI-1 siRNA transfection or administration of tiplaxtinin. Signaling pathways for EMT by smoking extract were through the phosphorylation of SMAD2 and ERK1/2, and finally Snail expression. Tiplaxtinin also suppressed the pulmonary fibrosis and PAI-1 expression in the bleomycin-induced rats.
CONCLUSION
Our data shows that CSE induces rat ATII cells to undergo EMT by PAI-1 via SMAD2-ERK1/2-Snail activation. This suppression of EMT by PAI-1 siRNA transfection or PAI-1 inhibitor in primary type II alveolar epithelial cells might be involved in the attenuation of bleomycin-induced pulmonary fibrosis in rats.

Keyword

Plasminogen Activator Inhibitor 1; Epithelial-Mesenchymal Transition; Pulmonary Fibrosis

MeSH Terms

Animals
Blotting, Western
Epithelial Cells
Epithelial-Mesenchymal Transition
Idiopathic Pulmonary Fibrosis
Indoleacetic Acids
Lung
Phosphorylation
Plasminogen
Plasminogen Activator Inhibitor 1
Plasminogen Activators
Polymerase Chain Reaction
Povidone
Pulmonary Fibrosis
Rats
Risk Factors
RNA, Small Interfering
Silicon Dioxide
Smoke
Smoking
Snails
Transfection
Indoleacetic Acids
Plasminogen
Plasminogen Activator Inhibitor 1
Plasminogen Activators
Povidone
RNA, Small Interfering
Silicon Dioxide
Smoke

Figure

  • Figure 1 Isolated rat alveolar type II (ATII) epithelial cells in phase contrast micrographs, ×200 (A) showing positive alkaline phosphatase staining, ×200 (B) and lamellar bodies at electron microscopy, bar=1µm (C).

  • Figure 2 Quantitative real time RT-PCR analysis of α-SMA (A), E-cadherin (B) and PAI-1 (C) in rat ATII cells. Total RNA was isolated from epithelial cells in each treatment group and subjected to quantitative RT-PCR using a iQ5 cycler instrument. CSE (5%) induced EMT was confirmed by decreasing the E-cadherin mRNA and increasing the α-SMA RNA. CSE also markedly increased PAI-1 mRNA expression. The PAI-1 siRNA transfection or 50µg of tiplaxtinin reversed the EMT and PAI-1 mRNA expression which were induced by CSE exposure on the rat ATII cells. Statistical analysis was performed using Mann-Whitney U test. CSE: cigarette smoking extract; EMT: epithelial-mesenchymal transition; RT-PCR: reverse transcription-polymerase.

  • Figure 3 Western blot analysis of PAI-1, α-SMA and E-cadherin in rat ATII cells. Rat alveolar epithelial cells were stimulated with CSE (5%), TGF-β (5 ng/mL) for 72 hours and total cell lysates were taken for immunoblot. Both CSE and TGF-β induced EMT was again confirmed by increased α-SMA expression and decreased E-cadherin expression. CSE also increased the PAI-1 expression. Transfection of PAI-1 siRNA or addition of tiplaxtinin (50µg) to the rat ATII cells reversed the CSE induced EMT and PAI-1 expression. Values given are the mean±SEM. Statistical analysis was performed using by Mann-Whitney U test. CSE: cigarette smoking extract; SEM: standard error of the mean.

  • Figure 4 Representative phase contrast images and immunofluorescene staining for E-cadherin (red) and α-SMA (green) expression with CSE, CSE plus PAI-1 siRNA transfection or tiplaxtinin treatment in primary rat type II epithelial cells; nuclei are stained with 4', 6-diamidino-2-phenylindole (blue). CSE induced EMT in type II epithelial cells were inhibited either by PAI-1 siRNA transfection or PAI-1 specific inhibitor, tiplaxtinin (Original magnification, ×200). CSE: cigarette smoking extract.

  • Figure 5 Representative phase contrast images and immunofluorescene staining for PAI-1 (red) and α-SMA (green) expression with CSE, CSE plus PAI-1 siRNA transfection or tiplaxtinin treatment in primary rat type II epithelial cells. CSE induced α-SMA and PAI-1 expression in type II epithelial cells were inhibited either by PAI-1 siRNA transfection or PAI-1 specific inhibitor, tiplaxtinin (Original magnification, ×200). CSE: cigarette smoking extract.

  • Figure 6 Effects of CSE and on TGF-β secretion of cultured rat ATII cells. Transfection of PAI-1 siRNA to the ATII cells or addition of tiplaxtinin (50µg) decreased the TGF-β concentrations in the supernatants of CSE-exposed rat ATII cells. CSE: cigarette smoking extract.

  • Figure 7 CSE induced the ERK1/2, SMAD2 phosphorylation and Snail expression in cultured rat ATII cells. ATII cell lysates were subjected to western blotting using the pERK1/2, pSmad2 and Snail antibodies. Blots were analyzed by densitometry. Transfection of PAI-1 siRNA to the ATII cells or addition of tiplaxtinin (50µg) decreased the CSE-induced ERK1/2, SMAD-2 phosphorylation and Snail expression. CSE: cigarette smoking extract.

  • Figure 8 Representative photomicrographs of rat lung tissue stained with a hematoxylin-eosin in control (A), bleomycin injury (B) and tiplaxtinin plus bleomycin (C). Severe pulmonary inflammation and fibrosis occurred after bleomycin injury on day 14 (B) and this histological change was markedly reduced by tiplaxtinin treatment (C). Lung fibrosis as evaluated by Ashcroft score was markedly reduced by tiplaxtinin treatment after bleomycin injury. The Ashcroft score are mean±SEM from six rats for each group. SEM: standard error of the mean. All pictures were taken at ×100 magnification.

  • Figure 9 The PAI-1 and TGF-β concentration in the BAL fluids were increased after intra-tracheal exposure to bleomycin to the rats and these increased levels were markedly decreased by tiplaxtinin treatment. PAI-1 and TGF-β concentrations were measured by ELISA at 14 days after exposure to bleomycin. Reported data are mean values±SEM from six rats for each group. ELISA: enzyme-linked immunosorbent assay; SEM: standard error of the mean.


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