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. 2016 Dec 23;6(2):215–222. doi: 10.1039/c6tx00378h

Cigarette smoke extract induces the epithelial-to-mesenchymal transition via the PLTP/TGF-β1/Smad2 pathway in RLE-6TN cells

Hong Chen a,†, Feng-ping Wu a,b,†, Yong-zhen Yang b, Xiu-ying Yu a, Lu Zhang a, Hui Zhang a, Ya-juan Chen a,✉
PMCID: PMC6060699  PMID: 30090492

graphic file with name c6tx00378h-ga.jpg Aim: The role of phospholipid transfer protein (PLTP) in the pathogenesis of the cigarette smoke extract (CSE)-induced epithelial-to-mesenchymal transition (EMT) has not been well described.

Abstract

Aim: The role of phospholipid transfer protein (PLTP) in the pathogenesis of the cigarette smoke extract (CSE)-induced epithelial-to-mesenchymal transition (EMT) has not been well described. In this study we investigated the effect of PLTP on the CSE-induced EMT of rat alveolar epithelial cells (RLE-6TN). Methods: The rats were exposed to air and cigarette smoke (CS) for 3 d and then the lungs were sectioned and examined using immunohistochemistry techniques. RLE-6TN cells were treated with different concentrations of CSE. PLTP siRNA was transfected into cells or SB431542 – an inhibitor of the transforming growth factor-β1 (TGF-β1) type I receptor – was administered prior to CSE exposure. The expression of EMT markers and PLTP was detected by qRT-PCR. The levels of PLTP, TGF-β1, p-Smad2, Smad2, and EMT proteins were analyzed by western blotting. Results: Lung injury and EMT were accompanied by up-regulation of PLTP and TGF-β1 in the CS-exposed rat model. EMT was induced by CSE in vitro, and the expression of PLTP, TGF-β1, and p-Smad2 was significantly increased after exposure to CSE (P < 0.05). Moreover, knockdown of PLTP and blocking of the TGF-β1/Smad2 pathway restrained the CSE-induced activation of the TGF-β1/Smad2 pathway and partly inhibited EMT by reversing E-cadherin expression and retarding the induction of N-cadherin and vimentin. In contrast, SB431542 had no effect on the expression of PLTP, while it ameliorated CSE-induced EMT. Conclusion: PLTP promotes the CSE-induced EMT process, in which the TGF-β1/Smad2 pathway is activated.

Introduction

The epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells lose their phenotypic characteristics and acquire mesenchymal features. EMT is increasingly recognized as a dynamic and reversible epigenetic reprogramming event that plays a role in embryogenesis, organ homeostasis in response to acute injury, or cancer progression/metastasis.1 Thus, we propose that EMT may be critically involved in the pathogenesis of lung injury.

Lung cells, particularly alveolar epithelial cells (AECs), are susceptible to stimuli such as cytokines and oxidants which are released into the environment.2 Several studies have indicated that cigarette smoke can induce EMT in the human non-small-cell lung carcinoma cell line (H358),3 human bronchial epithelial (HBE) cells,4 adenocarcinomic human alveolar epithelial cells (A549),5 and HSAEpiC cells;6 however, the underlying mechanism remains poorly studied.

Phospholipid transfer protein (PLTP) is a lipid metabolism-related glycoprotein, which is versatile and near ubiquitously-expressed and that is secreted in plasma7 and other fluid compartments,8 where PLTP regulates key biological processes.9,10The activity of PLTP is much higher in the plasma of smokers than in the plasma of non-smokers.11 Similarly, PLTP is further increased in patients with chronic obstructive pulmonary disease (COPD).12 We reported previously that PLTP participates in the regulation of transforming growth factor-β1 (TGF-β1) expression;13 however, the role of PLTP in the pathogenesis of CSE-induced EMT has not been well described. TGF-β1 is a major mediator of EMT and is the key cytokine in abnormal tissue remodeling.14,15 Our study demonstrated that CSE increases the expression of TGF-β1.16 In addition, a recently published study reported that high-mobility group box 1 (HMGB1) induces EMT via TGF-β1/Smad2/3 signaling in human A549 and rat RLE-6TN AECs.17 Smad2, as the downstream effector of TGF-β1, is activated by TGF-β1. For example, it has been reported that siRNA to Smad2 can partly abrogate TGF-β1-induced EMT.18 Hence, we assumed that PLTP may be implicated in CSE-induced EMT via the TGF-β1/Smad2 pathway in rat alveolar type II cells.

Materials and methods

Animals and animal exposure

Twenty male Wistar rats (weight = 180 ± 10 g) were purchased from the Chongqing Medical University Animal Center (Chongqing, China). All experiments were approved by the Chongqing Medical University Animal Care and Use Committee and were performed in compliance with the relevant animal care and use laws and institutional guidelines. The rats were exposed to air or whole-body CS (n = 10 per exposure) for 6 h per day on 3 consecutive days, using a modification of the method described by Ghio.19 The rats were sacrificed on d 4 and the lungs were photographed and fixed in 4% formalin.

Cells and reagents

RLE-6TN cells were acquired from the American Type Culture Collection (ATCC; no. CRL-2300; ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone Co., Logan, UT, USA), and continuously maintained at 37 °C under a 5% CO2 atmosphere. Rabbit monoclonal antibodies against PLTP, E-cadherin, and N-cadherin were purchased from Abcam, Inc. (Cambridge, MA, USA). The primary polyclonal antibody against TGF-β1 was obtained from ImmunoWay (Newark, DE, USA). Rabbit monoclonal antibodies against vimentin, Smad2, and p-Smad2 were obtained from Cell Signaling Technology, Inc. (Boston, MA, USA). The primary polyclonal antibody against GAPDH and the secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). RNA-max was purchased from Life Technologies (AB & Invitrogen, Carlsbad, CA, USA). SB431542 was obtained from Selleckchem (Houston, TX, USA).

CSE preparation

Research cigarettes (Hong Shen, China) were purchased from the Chongqing Tobacco Industry Co. (Chongqing, China). The composition of cigarettes was as follows: tar (11 mg per cigarette); carbon monoxide (17 mg per cigarette); and nicotine (1.1 mg per cigarette). CSE was prepared by bubbling smoke from 10 cigarettes into 10 ml DMEM without FBS according to the method described by Wirtz.20 Briefly, the smoke from one filtered cigarette was drawn through an experimental apparatus and completely combusted within 4 min. The smoke of 10 cigarettes was bubbled through 10 mL of DMEM without FBS. The liquid mixture was adjusted to a pH of 7.4 and filtered through a 0.22 μm Millipore filter (Millipore, Bedford, MA, USA) for sterilization. To standardize the CSE preparation, absorbance was measured at a wavelength of 320 nm using a Beckman DU 640 spectrophotometer (Fullerton, CA, USA) with DMEM as the blank. The absorbance (spectrogram) of CSE observed at 320 nm (approximately 1.36 ± 0.12) showed very little variation across different preparations. The concentration of the resulting solution was set as 100% and diluted to various concentrations according to the experimental design. The CSE solutions were always freshly prepared using exactly the same method and were used within 30 min after preparation.

Histology and immunohistochemistry (IHC) analyses

The right lung was embedded in paraffin and sectioned (4 μm) using standard procedures. The sections were stained with hematoxylin and eosin (H&E) to evaluate morphologic changes. In addition, the sections were immunostained using an indirect method with antibodies against PLTP, TGF-β1 (Santa Cruz Biotechnology Inc.), E-cadherin (ImmunoWay), N-cadherin, and vimentin, and visualization was performed with the avidin–biotin–peroxidase complex (ZhongShan Biotech, Beijing, China) method. Color development was performed with a DAB color development kit (ZhongShan Biotech).

Quantitative reverse transcription PCR (qRT-PCR)

Primers were purchased from Shanghai Bioengineering, Ltd (Shanghai, China). Total RNA was extracted with the TRIzol reagent (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer's instructions. Then, RNA was reverse-transcribed using the Takara PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Otsu). qRT-PCR was conducted in triplicate using SYBR Green Supermix according to the manufacturer's protocol (TaKaRa) with a mini real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Sequences of the primers used for qRT-PCR validation are as follows:

E-cadherin (5′-TCATCACAGACCCCAAGACC-3′; 5′-GATCTCCAGACCCACACCAA-3′);

N-cadherin (5′-AGATGACCCAAATGCCCTGA-3′; 5′-TGAAAGGCCGTAAGTGGGAT-3′);

vimentin (5′-TGACATTGAGATCGCCACCT-3′; 5′-TCATCGTGGTGCTGAGAAGT-3′); and

PLTP (5′-CGTGCGTAGTTCTGTGGATG-3′; 5′-CATCCTCTCGTCGTCATCCA-3′).

Western blotting analysis

Briefly, cells were extracted with cell lysis buffer. Lysates of cells (40 μg) were separated by SDS-PAGE and immunoblotted using antibodies against the following proteins: PLTP, TGF-β1, Smad2, p-Smad2, E-cadherin, N-cadherin, vimentin, and GAPDH. Chemiluminescence was performed to visualize the immunoreactivity of the protein. Quantity One software (Bio-Rad) was used to visualize and quantify the scanned figures.

Knockdown of PLTP

Shanghai GenePharma Co. (Shanghai, China) was entrusted to produce siRNA probes for PLTP and negative control (NC) scrambled siRNA. The cells were cultured in a 6-well plate for 24 h. Then, the PLTP or NC siRNA was transfected into cells by using RNA-max according to the manufacturer's instructions. Western blotting and qRT-PCR were used to examine PLTP silencing by siRNA at 48 h after transfection. Below is a list of PLTP siRNA:

PLTP (sense: 5′-GGACCUUCGAAGGUUUCAATT-3′; antisense: 5′-UUGAAACCUUCGAAGGUCCTT-3′).

Statistical analysis

Data are expressed as the mean ± SD. Statistical significance was determined by one-way analysis of variance followed by Dunnett's test or Student's t-test (GraphPad Prism, version 5.01, La Jolla, CA, USA). Differences were assumed to be statistically significant at P < 0.05.

Results

CS increases PLTP/TGF-β1 and induces EMT in the rat lung

After 3 days of CS exposure, the lung became congestive and edematous (Fig. 1a). As shown by H&E staining, alveolar walls became thin, lung tissue was destroyed, and alveolar spaces enlarged (Fig. 1b). The expression of PLTP (Fig. 1c) and TGF-β1 (Fig. 1d) in rat alveolar cells increased after exposure to CS. CS exposure significantly reduced E-cadherin (Fig. 1e) and increased N-cadherin (Fig. 1f) and vimentin (Fig. 1g) compared with controls.

Fig. 1. Cigarette smoke exposure activates PLTP/TGF-β1 and induces EMT in the rat lung. Male Wistar rats were randomly divided into a control group (n = 10) and a cigarette smoke (CS) exposure group (n = 10) and then exposed to air or CS for 6 hours per day on 3 consecutive days, lungs were removed under anesthesia on day 4. The fresh lung was photographed (a). H&E staining was performed to detect pathological changes (b). Expressions of PLTP (c), TGF-β1 (d) and EMT markers (e–g) were detected by IHC.

Fig. 1

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CSE induces EMT in lung epithelial RLE-6TN cells

After treatment with CSE at concentrations of 0.25%–1%, the level of expression of E-cadherin mRNA decreased in a dose-dependent manner (Fig. 2A), while CSE increased the mesenchymal marker, N-cadherin mRNA, in a dose-dependent manner (Fig. 2B). Furthermore, 1% CSE significantly increased the mRNA expression of vimentin (Fig. 2C). As shown in Fig. 2D, compared to the control, cells treated with CSE showed a significant decrease in E-cadherin and increase in N-cadherin and vimentin proteins. The changes in these proteins caused by CSE were dose-dependent, and at a concentration of 1% compared to the control group, CSE caused a 55% decrease in E-cadherin and a 157% and a 120% increase in N-cadherin and vimentin, respectively. This evidence suggests that CSE induced EMT in RLE-6TN cells. We therefore selected 1% of CSE to evaluate EMT markers in future experiments.

Fig. 2. CSE induces EMT in RLE-6TN cells. RLE-6TN cells were treated with CSE at concentrations of 0.25, 0.5 and 1% for 48 or 72 h. The mRNA expressions of E-cadherin (A), N-cadherin (B) and vimentin (C) were analyzed. The expressions of E-cadherin, N-cadherin and vimentin were detected by western blotting (D) and were quantified densitometrically (E). Values are the mean ± SD of three independent experiments. *P < 0.05 compared with the control. CSE, cigarette smoke extract.

Fig. 2

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CSE induces PLTP accumulation and TGF-β1/Smad2 activation in RLE-6TN cells

To determine the effect of PLTP and TGF-β1/Smad2 on CSE-induced EMT, cells were treated with different concentrations of CSE (0%, 0.25%, 0.5%, and 1.0%). Our results showed that CSE significantly enhanced the protein expression of PLTP in a dose-dependent manner (Fig. 3A and B). CSE significantly increased the expression of TGF-β1 and p-Smad2 at all doses, except the lowest CSE dose (0.25%), while there was no change in the expression of total Smad2, indicating that PLTP and TGF-β1/Smad2 signaling might be involved in CSE-induced EMT.

Fig. 3. CSE up-regulates PLTP, TGF-β1 and p-Smad2 expressions. Rat alveolar epithelial cells were treated with 0.25–1% CSE for 72 h, and the expressions of PLTP, TGF-β1, p-Smad2, Smad2 and GAPDH in cell lysates were detected by western blotting (A) and were quantified by densitometry (B). Values are the mean ± SD of three independent experiments. *P < 0.05 compared with the control. CSE, cigarette smoke extract.

Fig. 3

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PLTP silencing inhibited CSE-induced EMT, TGF-β1 expression, and Smad2 phosphorylation in RLE-6TN cells

To further determine whether or not PLTP is required for CSE-induced EMT in RLE-6TN cells, an siRNA that targets PLTP mRNA was prepared according to our previous study.16 As shown, PLTP siRNA resulted in an 80% reduction in the expression of PLTP mRNA (Fig. 4A), and a 65% reduction in the level of PLTP protein (Fig. 4B). In agreement with another study, CSE triggered Smad2 phosphorylation, increased the expression of PLTP, TGF-β, N-cadherin and vimentin, and decreased the expression of E-cadherin (Fig. 4C–G). In contrast, the knockdown of PLTP by siRNA significantly prevented EMT in response to CSE, reducing the increased mesenchymal markers and elevating the decreased epithelial markers (Fig. 4F and G). Furthermore, PLTP and activation of TGF-β1 and p-Smad2 in response to CSE stimulation were strongly attenuated with the deficiency of PLTP (Fig. 4C and D), suggesting that CSE-induced EMT requires the activation of PLTP and PLTP acts in the signaling pathway of TGF-β1/Smad2 regulated by CSE.

Fig. 4. Cells were treated with siRNA for PLTP and negative control scrambled siRNA (NC) at a concentration of 60 pmol for 48 or 72 h. The expression of PLTP was analyzed by qRT-PCR (A) and western blotting (B). After the siRNA was transfected into cells, the cells were exposed to CSE. The expression of PLTP was analyzed by qRT-PCR (C). The expressions of PLTP, TGF-β1 and p-Smad2 were detected by western blotting (D) and were quantified densitometrically (E); EMT markers were detected by western blotting (F) and were quantified by densitometry (G). Values are the mean ± SD of three independent experiments. *P < 0.05, significant difference compared to the NC group; #P < 0.05, significant difference compared to the NC + CSE group. CSE, cigarette smoke extract; NC, negative control scrambled siRNA. siRNA, PLTP-siRNA. CSE, cigarette smoke extract.

Fig. 4

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PLTP is an upstream signaling molecule of the TGF-β1/Smad2 pathway through which CSE induces EMT in RLE-6TN cells

Both PLTP and TGF-β1/Smad2 are involved in CSE-induced EMT; therefore it is important to evaluate whether PLTP and TGF-β1/Smad2 have an independent effect on CSE-induced EMT. SB431542 was used to block the TGF-β1/Smad2 pathway. To detect the efficiency of suppression by SB431542, different concentrations of SB431542 were tested by western blotting to filter the optimal concentration (Fig. 5A). As shown, 1000 nmol L–1 of SB431542 resulted in a 58% reduction in the level of p-Samd2 protein (Fig. 5A and B). In contrast, the expression of PLTP was not significantly affected by SB431542. Thus, 1000 nmol L–1 of SB431542 was added to the cell culture, and subsequently exposed to CSE. As shown in Fig. 5C, SB431542 clearly inhibited CSE-induced activation of p-Smad2 and abrogated EMT. Knockdown of PLTP reduced the CSE-induced activation of the TGF-β1/Smad2 pathway and inhibited EMT (Fig. 4D–G); however, SB431542 had no effect on the expression of PLTP (Fig. 5A–D). Overall, these results suggest that PLTP participates in CSE-induced EMT, acting as an upstream signaling molecule of the TGF-β1/Smad2 signaling pathway.

Fig. 5. SB431542 was used to block the TGF-β1/Smad2 pathway and the expressions of PLTP, p-Smad2 and EMT markers were analyzed by western blotting. Cells were starved for 8 h and treated with 10–1000 nmol L–1 SB431542 for 72 h. The expressions of PLTP and p-Smad2 were analyzed (A) and quantified densitometrically (B). RLE-6TN cells starved and pretreated with or without SB431542 (1000 nmol L–1, 1 h) were exposed to CSE for 72 h. The expressions of PLTP, p-Smad2 and EMT markers were analyzed (C) and the relative expression ratio was quantified by densitometry (D). Values are the mean ± SD of three independent experiments. *P < 0.05, significant difference compared to the control group; #P < 0.05, significant difference compared to the CSE-induced groups. CSE, cigarette smoke extract.

Fig. 5

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Discussion

Cigarette smoking is a major cause of morbidity and mortality. Exposure to CSE can lead to a large number of diseases, such as lung cancer,21 idiopathic pulmonary fibrosis,22 COPD,22,23 and lung injury.24 Studies have shown that CSE exposure may be a risk factor of acute lung injury.25–28 Some studies have shown that CSE can promote the EMT process in the lung.4,6,29 In agreement with this notion, a recent study reported that EMT occurred after exposure to CSE, as measured by changes in E-cadherin, N-cadherin, and vimentin in H358.3 A study has shown that CS can promote the EMT process in A549 due to a decrease in E-cadherin and elevation of a-SMA.5 Bartis and colleagues30 have reported that CSE induces the EMT process in differentiated human bronchial epithelial cells. These findings are all consistent with the findings in our study. Interestingly, we failed to detect CSE-induced EMT in HBE cells. Indeed, exposure to CSE for 72 h decreased E-cadherin, but did not increase N-cadherin or vimentin, which has not been previously reported. After treatment with CSE at concentrations of 0.25%–1%, the level of expression of E-cadherin mRNA decreased at all doses, except the lowest CSE dose (0.25%). However, there is no difference in the expressions of N-cadherin and vimentin after CSE exposure. In contrast with our assumption, one reason for this unexpected observation may be inadequate exposure time. Several studies have shown that chronic exposure of HBE cells to CSE for approximately 40 passages induces EMT.29,31,32 In addition, it is likely that this discrepancy is caused by differences in cell sources. HBE, which underwent EMT after 72 h of CSE exposure, was isolated and cultured from human lung tissue;4,33,34 however, our HBE was not primary. Considering that secondhand CS is a common environmental hazard35,36 and given the less-than-complete success of smoking cessation efforts, it is important to gain an understanding of how CS affects lung diseases so that effective strategies can be developed to prevent and treat CS-related diseases.

The lung is the dominant site expressing PLTP mRNA in humans and mice,37,38 suggesting that this protein might have an important role in maintaining normal functions of this organ. It has been reported that plasma PLTP activity is much higher in smokers compared with non-smokers.11 Moreover, recent studies have indicated that EMT is induced by cigarette smoke in bronchial epithelial cells;3,4,39 however, these results are not sufficient to conclude that CSE induces EMT through PLTP. Our work indicated that lung injury happens (Fig. 1a and b) and PLTP and TGF-β1 expression increases (Fig. 1c and d) in lung tissue after CS exposure; meanwhile, EMT occurs (Fig. 1e–g). Importantly, CSE also induces EMT (Fig. 2) and increases PLTP and TGF-β1 (Fig. 3) in vitro. EMT can be partially inhibited by PLTP knockdown (Fig. 4). These results show that PLTP plays a role in CSE-induced EMT of rat alveolar type II cells. Nevertheless, CSE decreased the expression of PLTP in HBE (data not shown), which is inconsistent with studies conducted in RLR-6IN. It is tempting to speculate that the increase in PLTP in RLR-6IN plays a role in regulating alveolar surfactant metabolism. Alternatively, induction of PLTP expression might simply be a consequence of surfactant deficiency, which leads to micro-atelectasis and attendant hypoxia. Cigarette smoking reduces the amount of surfactant on the alveolar surface,15 but has been shown to increase the number of alveolar type II epithelial cells.16 We speculate that type II cell hyperplasia can also stimulate PLTP production to transport more surfactant to the alveolar membrane; however, as yet there has been limited research focusing on the relationship between CSE and PLTP in HBE cells. It is possible that the decrease in PLTP may be due to an inability of the cells to synthesize pulmonary surfactant.

TGF-β1 plays a pivotal role in the pathogenesis of EMT.15,18 Kasai18 has concluded that TGF-β1-induced EMT occurs through the phosphorylation of Smad2 and investigated the critical role of Smad2 in TGF-β-induced EMT in A549 cells. A recent study demonstrated that CSE increases the expression of TGF-β1 and activates Smad2 in pulmonary epithelial cells.40 Our previous study revealed that PLTP is involved in the oxidized-low density lipoprotein (Ox-LDL)-induced production of TGF-β1.13 The finding was confirmed, in our current research, that CSE increases the expression of PLTP and TGF-β1 (Fig. 3). These results imply that the activation of the TGF-β1/Smad2 pathway by CSE may depend on PLTP. In our present work, we demonstrated that PLTP siRNA inhibits the production of TGF-β1 and p-Smad2 induced by CSE (Fig. 4). There was no significant difference in the expression of PLTP after blocking the TGF-β1/Smad2 pathway with SB431542 (Fig. 5). Taken together, these data suggest that PLTP promotes the CSE-induced EMT process, in which the TGF-β1/Smad2 pathway is activated. TGF-β1 increased after CSE exposure in HBE. Nevertheless, TGF-β1 showed no significant change, and HBE requires high concentrations or a suitable exposure time.

Conclusions

In conclusion, the present study has demonstrated that CSE up-regulates PLTP production and activates the TGF-β1/Smad2 pathway while inducing EMT in RLE-6TN cells. PLTP promotes CSE-induced EMT, acting as the upstream signaling molecule of the TGF-β1/Smad2 pathway. Understanding the mechanism of CSE-induced EMT may help us understand the molecular mechanisms involved in the pathogenesis of lung diseases. Our study provides new insights into the fundamental basis of EMT and suggests a possible new course of therapy for lung diseases.

Authors contribution

Hong Chen, Feng-ping Wu and Ya-juan Chen designed the research; Hong Chen, Feng-ping Wu and Xiu-ying Yu performed the research; Lu Zhang and Hui Zhang contributed new reagents or analytic tools; Feng-ping Wu and Yong-zhen Yang analyzed the data; Hong Chen, Feng-ping Wu and Ya-juan Chen contributed to the writing of the paper.

Conflict of interest

There are no conflicts of interest to declare.

Acknowledgments

This work was supported by the Natural Science Foundation for the Youth (NSFY) of China (No. 81200009).

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