D-4F, an apolipoprotein A-I mimetic, inhibits TGF-β1 induced epithelial-mesenchymal transition in human alveolar epithelial cell

https://doi.org/10.1016/j.etp.2016.07.005Get rights and content

Abstract

Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10 ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.

Introduction

Epithelial-mesenchymal transition (EMT) is crucial to a number of important biological events including embryogenesis, development, tissue remodeling, cancer progression, and organ fibrosis (Kalluri and Weinberg, 2009). Pulmonary fibrosis is particularly widespread and has always been a major threat to mankind. The irreversible fibrosis may lead to gradual lung deterioration, and there is currently no effective cure (King et al., 2000, Meltzer and Noble, 2008). As a dysregulated and over-active wound-healing biosynthetic process, pulmonary fibrosis can be induced by chemicals, physical particles, infections, ischemia, autoimmune attacks, or idiopathic factors. Although preservations of lung function were reported in idiopathic pulmonary fibrosis (IPF) patients and several anti-fibrotic agents were demonstrated to prevent fibrosis from progression, no clinical available pharmaceutical has been developed to resolve fibrotic nodules and reverse established fibrosis (Choe et al., 2010, Demedts et al., 2005, Iyer et al., 1998, Kao et al., 2013, Pini et al., 2010).

Recently, a high-throughput whole-proteome study revealed that the concentration of apolipoprotein A-I (Apo A-I) was greatly reduced in the bronchoalveolar lavage (BAL) fluid of IPF patients (Kim et al., 2010). In addition, intranasal administering of Apo A-I reduced the number of inflammatory cells and collagen deposition in bleomycin induced pulmonary fibrosis model, indicating that Apo A-I may be effective against experimental lung injury and fibrosis (Kim et al., 2010). However, obstacles in protein delivery and absorption inside the narrow airways limited the efficacy of intranasal Apo A-I treatment. Consequently, investigations were performed on transgenic mice expressing human Apo A-I, and resolvation of established pulmonary fibrosis was reported for the first time ever (Lee et al., 2013). In the silica-induced fibrosis model, the number of inflammatory cells, fibrotic nodule volume, collagen deposition and TGF-β1 concentration were all decreased when Apo A-I local expression was induced in transgenic mice, suggesting that overexpressed Apo A-I was able to reduce lung inflammation and reverse the established fibrosis (Lee et al., 2013). Exciting as it is, Apo A-I gene therapy still has a long way to go. Thus, in the battle against pulmonary fibrosis, a more applicable and readily available substitute for Apo A-I is called for.

D-4F is a polypeptide originally synthesized to mimic the functions of Apo A-I in order to treat cardiovascular disorders (Navab et al., 2002, Navab et al., 2003). Consisting of 18 D-amino acids, D-4F is not recognized by mammalian peptidases, providing remarkable pharmacokinetic properties (Getz and Reardon, 2011, Navab et al., 2005b). Multiple studies suggested that D-4F not only inhibited atherosclerosis in animal model, but also safely improved anti-inflammatory index in clinical trials on patients with coronary disease (Bloedon et al., 2008, Navab et al., 2006, Navab et al., 2002, Navab et al., 2005a, Navab et al., 2004). Interestingly, D-4F was also reported to decrease airway hyper-responsiveness, oxidative stress, and inflammation in asthmatic model, implying that D-4F might be utilized to treat asthma (Nandedkar et al., 2011). Nevertheless, the cellular mechanism for the effects of D-4F on respiratory system has never been investigated.

The pathogenesis of pulmonary fibrosis and asthmatic airway remodeling is generally believed to be associated with EMT (Boxall et al., 2006, Selman and Pardo, 2003, Zavadil and Böttinger, 2005). EMT is defined as the phenotypic and functional transition from polarized epithelial cells to fibroblast-resembling mesenchymal cells, with the loss of interaction with basement membrane, gain of migratory capacity, and production of excess extracellular matrix (ECM) (Kalluri and Weinberg, 2009). The initiation of EMT is closely associated with transforming growth factor β1 (TGF-β1), and the development of EMT is accompanied by the down-regulation of epithelial marker E-cadherin and the up-regulation of mesenchymal marker vimentin (Rastaldi et al., 2002, Yang et al., 2002). Numerous evidences suggest that TGF-β1 is a pro-fibrotic cytokine and TGF-β1 induced EMT plays a key role in pulmonary fibrosis and asthma (Holgate et al., 2003, Kasai et al., 2005, Mutlu et al., 2012, Willis et al., 2005).

Therefore, this study investigated the effects of D-4F on TGF-β1 induced EMT. A well validated model of EMT in human type II alveolar epithelial cell line A549 was recruited. Cellular morphological transitions, transcriptional and translational changes in epithelial and mesenchymal makers were characterized when the TGF-β1 induced EMT was treated with different concentrations of D-4F. The reported results demonstrate that D-4F prevents TGF-β1 induced morphological changes in A549, and inhibits TGF-β1 induced regulations of EMT markers. Based on these results, we believe that D-4F may perform anti-fibrotic and anti-asthmatic functions by inhibiting alveolar EMT, thereby supporting future clinical application of D-4F in treating pulmonary fibrosis and asthma.

Section snippets

Reagents

Human transforming growth factor β1 (TGF-β1), phenylmethylsulfonyl fluoride (PMSF), diethylpyrocarbonate (DEPC), l-glutamine, and penicillin-streptomycin were obtained from Sigma-Aldrich (St. Louis, MO). Anti-E-cadherin, anti-vimentin and anti-β-actin rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-rabbit IgG secondary antibody (horseradish peroxidase) was bought from Novus Biologicals (Minneapolis, MN). Trizol was purchased from Invitrogen (Carlsbad, CA).

Characterization of TGF-β1 induced EMT in A549 cells

In order to establish a valid EMT model and to characterize the effects of TGF-β1 induced EMT in A549 cells, evaluations of cellular morphology and EMT marker expression were performed at various time lengths. A549 cells were synchronized in the serum free medium for 24 h and then exposed to 10 ng/ml of TGF-β1 or the control. Upon TGF-β1 stimulation, photographs of A549 cells were taken at 0, 24, and 48 h, respectively. In the presence of TGF-β1, A549 cells assumed an elongated spindle shape with

Discussion

The major finding of the present study was that D-4F inhibited TGF-β1 induced EMT in human alveolar epithelial cells. D-4F was able to prevent the fibroblast-like phenotype transition in TGF-β1 treated A549 cells, and preserve the epithelial morphologies, including cell shape, size and cellular contacts. In addition, D-4F was able to significantly suppress the TGF-β1 induced down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. When TGF-β1 treated

Grants

This work was supported by the National Nature Science Foundation of China (Grant 81402642 and 81502768), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry, to Jingyuan Xiong), and the Science and Technology Department of Sichuan Province (Grant 2015JY0212).

Acknowledgments

The authors would like to thank professor Xiaofang Pei for her generous support throughout this study. We would also like to thank Dr. Liantian Tian, Yuqin Yao and Dr. Ying Shi for their helpful discussions regarding this manuscript.

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