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Methotrexate-Induced Epithelial–Mesenchymal Transition in the Alveolar Epithelial Cell Line A549

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Abstract

Purpose

Methotrexate (MTX) therapy of certain cancers and rheumatoid arthritis often induces serious interstitial lung complications including pulmonary fibrosis. In this study, we investigated the epithelial–mesenchymal transition (EMT) induced by MTX and by transforming growth factor (TGF)-β1 in the human alveolar epithelial cell line A549 in order to develop new strategies for the prevention of EMT.

Methods

First, we examined the effect of TGF-β1 and MTX on cell morphology and the expression of EMT-related mRNAs in A549 cells. Then, the effects of SB431542 (SB), a potent inhibitor of TGF-β receptor kinase, and a neutralizing antibody for TGF-β1 on the phenotypic changes of A549 cells induced by TGF-β1 and MTX were examined.

Results

After incubation with TGF-β1 and MTX, the mRNA expression of epithelial markers such as cytokeratin 19 was reduced, while that of mesenchymal markers such as α-smooth muscle actin was increased. SB suppressed the development of morphological changes and partially rescued alterations in mRNA expression of EMT markers induced by MTX. In addition, the enhancement of SMAD2 phosphorylation by MTX was also prevented by SB. On the other hand, EMT-related changes induced by MTX were not affected by a neutralizing antibody for TGF-β1.

Conclusion

We have demonstrated that phenotypic changes of A549 cells induced by MTX are partly mediated by a TGF-β1-related intracellular signaling pathway, although TGF-β1 itself is not directly involved in this process.

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Abbreviations

CK19:

Cytokeratin 19

EMT:

Epithelial–mesenchymal transition

MTX:

Methotrexate

SB:

SB431542

α-SMA:

α-smooth muscle actin

TGF-β1:

Transforming growth factor-β1

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

References

  1. Chattopadhyay S, Moran RG, Goldman ID (2007) Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther 6:404–417

    Article  CAS  PubMed  Google Scholar 

  2. Cronstein BN (2006) Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacol Rev 57:163–172

    Article  Google Scholar 

  3. Niehues T, Lankisch P (2006) Recommendations for the use of methotrexate in juvenile idiopathic arthritis. Paediatr Drugs 8:347–356

    Article  PubMed  Google Scholar 

  4. Calvo I, Anton J, Lopez Robledillo JC et al. (2015) Recommendations for the use of methotrexate in patients with juvenile idiopathic arthritis. An Pediatr (Barc)

  5. Kim KK, Kugler MC, Wolters PJ et al (2006) Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 103:13180–13185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Thiery JP, Acloque H, Huang RY et al (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139:871–890

    Article  CAS  PubMed  Google Scholar 

  7. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 15:178–196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moeller A, Ask K, Warburton D et al (2008) The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J Biochem Cell Biol 40:362–382

    Article  CAS  PubMed  Google Scholar 

  9. Wynn TA (2011) Integrating mechanisms of pulmonary fibrosis. J Exp Med 208:1339–1350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gonzalez DM, Medici D (2014) Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 7:re8

    Article  PubMed  PubMed Central  Google Scholar 

  11. Massague J (1998) TGF-beta signal transduction. Annu Rev Biochem 67:753–791

    Article  CAS  PubMed  Google Scholar 

  12. Mei Q, Saiz L (2014) Literature-based automated reconstruction, expansion, and refinement of the TGF-beta superfamily ligand-receptor network. J Membr Biol 247:381–386

    Article  CAS  PubMed  Google Scholar 

  13. Massague J (2008) TGFbeta in Cancer. Cell 134:215–230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Attisano L, Lee-Hoeflich ST (2001) The Smads. Genome Biol 2: Reviews 3010

  15. Willis BC, Borok Z (2007) TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 293:L525–L534

    Article  CAS  PubMed  Google Scholar 

  16. Chen LJ, Ye H, Zhang Q et al (2015) Bleomycin induced epithelial-mesenchymal transition (EMT) in pleural mesothelial cells. Toxicol Appl Pharmacol 283:75–82

    Article  CAS  PubMed  Google Scholar 

  17. Ohbayashi M, Kubota S, Kawase A et al (2014) Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis. J Toxicol Sci 39:319–330

    Article  CAS  PubMed  Google Scholar 

  18. Takano M, Yamamoto C, Yamaguchi K et al (2015) Analysis of TGF-beta1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3. Drug Metab Pharmacokinet 30:111–118

    Article  CAS  PubMed  Google Scholar 

  19. Kasai H, Allen JT, Mason RM et al (2005) TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT). Respir Res 6:56

    Article  PubMed  PubMed Central  Google Scholar 

  20. Kim JH, Jang YS, Eom KS et al (2007) Transforming growth factor beta1 induces epithelial-to-mesenchymal transition of A549 cells. J Korean Med Sci 22:898–904

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kawami M, Miyamoto M, Yumoto R et al (2015) Methotrexate influx via folate transporters into alveolar epithelial cell line A549. Drug Metab Pharmacokinet 30:276–281

    Article  CAS  PubMed  Google Scholar 

  22. Cronstein BN (1996) Molecular therapeutics. Methotrexate and its mechanism of action. Arthritis Rheum 39:1951–1960

    Article  CAS  PubMed  Google Scholar 

  23. Lynch JP 3rd, McCune WJ (1997) Immunosuppressive and cytotoxic pharmacotherapy for pulmonary disorders. Am J Respir Crit Care Med 155:395–420

    Article  PubMed  Google Scholar 

  24. van Ede AE, Laan RF, Blom HJ et al (1998) Methotrexate in rheumatoid arthritis: an update with focus on mechanisms involved in toxicity. Semin Arthritis Rheum 27:277–292

    Article  PubMed  Google Scholar 

  25. Kalluri R, Neilson EG (2003) Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 112:1776–1784

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Iwano M, Plieth D, Danoff TM et al (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110:341–350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bonniaud P, Margetts PJ, Kolb M et al (2005) Progressive transforming growth factor beta1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Med 171:889–898

    Article  PubMed  Google Scholar 

  28. Higashiyama H, Yoshimoto D, Kaise T et al (2007) Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis. Exp Mol Pathol 83:39–46

    Article  CAS  PubMed  Google Scholar 

  29. Inman GJ, Nicolas FJ, Callahan JF et al (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62:65–74

    Article  CAS  PubMed  Google Scholar 

  30. Koyama S, Sato E, Takamizawa A et al (2003) Methotrexate stimulates lung epithelial cells to release inflammatory cell chemotactic activities. Exp Lung Res 29:91–111

    Article  CAS  PubMed  Google Scholar 

  31. Kim YJ, Song M, Ryu JC (2009) Inflammation in methotrexate-induced pulmonary toxicity occurs via the p38 MAPK pathway. Toxicol 256:183–190

    Article  CAS  Google Scholar 

  32. Wu D, Cheng J, Sun G et al (2016) p70S6 K promotes IL-6-induced epithelial-mesenchymal transition and metastasis of head and neck squamous cell carcinoma. Oncotarget. doi:10.18632/oncotarget.9282

    Google Scholar 

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Acknowledgments

We thank the Analysis Center of Life Science of the Hiroshima University for the opportunity to use their facilities. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences (JSPS) (NOs; 26293033, 15K08074) and by a Grant for Exploratory Research of Young Researchers from Hiroshima University.

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Authors and Affiliations

  1. Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734-8553, Japan

    Masashi Kawami, Rika Harabayashi, Mioka Miyamoto, Risako Harada, Ryoko Yumoto & Mikihisa Takano

Authors
  1. Masashi Kawami
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  2. Rika Harabayashi
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  3. Mioka Miyamoto
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  4. Risako Harada
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  5. Ryoko Yumoto
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  6. Mikihisa Takano
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Corresponding author

Correspondence to Mikihisa Takano.

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The authors declare that there are no conflicts of interest.

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Kawami, M., Harabayashi, R., Miyamoto, M. et al. Methotrexate-Induced Epithelial–Mesenchymal Transition in the Alveolar Epithelial Cell Line A549. Lung 194, 923–930 (2016). https://doi.org/10.1007/s00408-016-9935-7

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  • DOI: https://doi.org/10.1007/s00408-016-9935-7

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