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

  • Molecular Toxicology
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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.

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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

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Acknowledgments

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

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Authors and Affiliations

  1. Department of Respiratory Medicine, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Yangpu District, Shanghai, 200433, People’s Republic of China

    Yunye Ning, Haidong Huang, Yuchao Dong, Qinying Sun, Wei Zhang, Wujian Xu & Qiang Li

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  1. Yunye Ning
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  2. Haidong Huang
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  3. Yuchao Dong
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  4. Qinying Sun
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  5. Wei Zhang
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  6. Wujian Xu
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  7. Qiang Li
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Corresponding author

Correspondence to Qiang Li.

Additional information

Y. Ning, H. Huang and Y. Dong contributed equally to the paper.

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Ning, Y., Huang, H., Dong, Y. et al. 5-Aza-2′-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching. Arch Toxicol 87, 871–881 (2013). https://doi.org/10.1007/s00204-012-1008-y

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  • DOI: https://doi.org/10.1007/s00204-012-1008-y

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