Archives of Toxicology

, Volume 87, Issue 5, pp 871–881 | Cite as

5-Aza-2′-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching

  • Yunye Ning
  • Haidong Huang
  • Yuchao Dong
  • Qinying Sun
  • Wei Zhang
  • Wujian Xu
  • Qiang Li
Molecular Toxicology

Abstract

Airway smooth muscle (ASM) cell phenotypic switching played an important role in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell phenotypic switching from a mature to pro-remodeling phenotype, but the mechanism remained incompletely understood. This study was to explore the effect of DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (Aza-CdR) on PDGF-induced rat ASM cell phenotypic switching and biological behaviors. Rat airway smooth muscle (RASM) cells were obtained by primary explant techniques. Western blot, 3-dimensional gel contraction, transwell and wound healing assay, and MTT were applied to detect cell phenotypic switching, contractility, migration and proliferation, respectively. Cytoskeleton rearrangement was observed by immunofluorescence. Results showed Aza-CdR inhibited PDGF-induced down-regulation of contractile markers in RASM cells and increased cell contractility. Aza-CdR inhibited PDGF-induced RASM cell migration by abrogating cell morphology change and cytoskeletal reorganization and attenuated the effect of PDGF on proliferating cell nuclear antigen expression and cell cycle progression, ultimately cell proliferation. PDGF-induced DNA methyltransferase 1 (DNMT1) expression was mediated by activation of PI3K/Akt and ERK signaling in RASM cells. Selective depletion of DNMT1 protein by Aza-CdR inhibited PDGF-induced RASM cell phenotypic switching, revealing DNMT1-mediated DNA methylation was implicated in asthmatic ASM remodeling. We proposed for the first time that DNMT1 played a key role in PDGF-induced RASM cell phenotypic switching and Aza-CdR is promising in intervening ASM remodeling in asthma. Although study of abnormal DNA methylation in PDGF-stimulated ASM cells is in its infancy, this work contributes to providing new insights into the mechanism of ASM remodeling and may be helpful for developing effective treatments for airway remodeling in asthma.

Keywords

5-Aza-2′-deoxycytidine Airway smooth muscle cells Phenotypic switching DNA methylation DNA methyltransferase 1 

Abbreviations

RASMC

Rat airway smooth muscle cell

PDGF

Platelet-derived growth factor

Aza-CdR

5-Aza-2′-deoxycytidine

DNMT1

DNA methyltransferase 1

PCNA

Proliferating cell nuclear antigen

OD

Optical density

MRLC

Myosin regulatory light chain

MLCK

Myosin light-chain kinase

2-D

Two-dimensional

3-D

Three-dimensional

TBST

Tris-buffered saline with 0.1 % Tween-20

PI3K

Phosphatidylinositol 3-kinase

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China [Grant No. 81100012, 81170060].

Conflict of interest

None.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Yunye Ning
    • 1
  • Haidong Huang
    • 1
  • Yuchao Dong
    • 1
  • Qinying Sun
    • 1
  • Wei Zhang
    • 1
  • Wujian Xu
    • 1
  • Qiang Li
    • 1
  1. 1.Department of Respiratory Medicine, Changhai HospitalSecond Military Medical UniversityYangpu District, ShanghaiPeople’s Republic of China

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