Toxicology Letters 226 (2014) 150–162 Contents lists available at ScienceDirect Toxicology Letters j o ur na l ho me page: www.elsevier.com/locate/toxlet Epithelial–mesenchymal transition involved in pulmonary fibrosis induced by multi-walled carbon nanotubes via TGF-beta/Smad signaling pathway Tian Chen a , Haiyu Nie b , Xin Gao a , Jinglin Yang a , Ji Pu a , Zhangjian Chen a , Xiaoxing Cui a , Yun Wang a , Haifang Wang c , Guang Jia a,∗ a Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, No. 38 Xueyuan Rd, Beijing 100191, China b Department of Applied Chemistry, China Agricultural University, No. 2 Yuanmingyuan West Rd, Beijing 100194, China c Institute of Nanochemistry and Nanobiology, Shanghai University, No. 99 Shangda Rd, Shanghai 200444, China h i g h l i g h t s • MWCNT induced pulmonary fibrosis in a length dependent manner. • Only long MWCNT produced the observed pulmonary fibrosis in C57Bl/6J mice. • EMT contributed to the adverse out- come of pulmonary fibrosis induced by MWCNT. • TGF-/p-Smad2 signaling pathway played a pivotal role in the occur- rence of EMT. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 6 December 2013 Received in revised form 27 January 2014 Accepted 3 February 2014 Available online 12 February 2014 Keywords: MWCNT Pulmonary fibrosis Epithelial–mesenchymal transition TGF- Smad a b s t r a c t Multi-walled carbon nanotubes (MWCNT) are a typical nanomaterial with a wide spectrum of commercial applications. Inhalation exposure to MWCNT has been linked with lung fibrosis and mesothelioma-like lesions commonly seen with asbestos. In this study, we examined the pulmonary fibrosis response to different length of MWCNT including short MWCNT (S-MWCNT, length = 350–700 nm) and long MWCNT (L-MWCNT, length = 5–15 m) and investigated whether the epithelial–mesenchymal transition (EMT) occurred during MWCNT-induced pulmonary fibrosis. C57Bl/6J male mice were intratracheally instilled with S-MWCNT or L-WCNT by a single dose of 60 g per mouse, and the progress of pulmonary fibro- sis was evaluated at 7, 28 and 56 days post-exposure. The in vivo data showed that only L-MWCNT increased collagen deposition and pulmonary fibrosis significantly, and approximately 20% of pro- surfactant protein-C positive epithelial cells transdifferentiated to fibroblasts at 56 days, suggesting the occurrence of EMT. In order to understand the mechanism, we used human pulmonary epithelial cell line A549 to investigate the role of TGF-/p-Smad2 signaling pathway in EMT. Our results showed that L- MWCNT downregulated E-cadherin and upregulated -smooth muscle actin (-SMA) protein expression in A549 cells. Taken together, both in vivo and in vitro study demonstrated that respiratory exposure to MWCNT induced length dependent pulmonary fibrosis and epithelial-derived fibroblasts via TGF-/Smad pathway. © 2014 Elsevier Ireland Ltd. All rights reserved. ∗ Corresponding author. Tel.: +86 10 8280 2333; fax: +86 10 8280 2333. E-mail addresses: chentian@bjmu.edu.cn (T. Chen), niehaiyu342@163.com (H. Nie), gaoxin0 0@163.com (X. Gao), goodboylinlin@163.com (J. Yang), puji328@bjmu.edu.cn (J. Pu), czjczj01@163.com (Z. Chen), cuixiaoxing@gmail.com (X. Cui), wangyun@bjmu.edu.cn (Y. Wang), hwang@shu.edu.cn (H. Wang), jiaguangjia@bjmu.edu.cn (G. Jia). http://dx.doi.org/10.1016/j.toxlet.2014.02.004 0378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.