Role of integrin α2 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells.

Methotrexate (MTX) is widely used to treat various diseases. However, it induces adverse reactions like serious lung injury, including pulmonary fibrosis. Increasing evidence suggests that epithelial-mesenchymal transition (EMT) in injured alveolar epithelium contributes to the development of the pathophysiological state of the lung. We demonstrated that MTX induced EMT in cultured alveolar epithelial cell lines. Integrin-mediated signaling is considered a significant factor in recognizing the EMT process. However, the relationship between MTX-induced EMT and integrin family members is poorly understood. In the present study, we aimed to clarify the role of integrin in MTX-induced EMT in A549 and NCI-H1299 (H1299) cells by focusing on the integrin alpha 2 (ITGA2) subunit, selected based on our microarray analysis. MTX treatment for 72 h significantly increased the mRNA and cell surface expression of ITGA2 in both cell lines. However, this upregulation by MTX was suppressed by co-treatment with SB431542 and folic acid, which are inhibitors of MTX-induced EMT in A549 cells. The mRNA expression levels of EMT-related genes were more affected in the MTX-treated A549 cells with high ITGA2 expression than in those with low ITGA2 expression. Finally, E7820, an ITGA2 inhibitor, suppressed MTX-induced EMT-related phenotypic changes, such as morphology and mRNA and protein expression of α-smooth muscle actin, a representative EMT marker. These findings suggest that ITGA2 may play a key role in MTX-induced EMT in alveolar epithelial cells.

Role of Nrf2 in Methotrexate-Induced Epithelial-Mesenchymal Transition in Alveolar A549 Cells.

Methotrexate (MTX) is known to induce serious lung diseases, such as pulmonary fibrosis. Although we demonstrated that MTX is associated with epithelial-mesenchymal transition (EMT), the underlying mechanism remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative stress response regulator, is related to EMT induction. In the present study, we examined the association of Nrf2 with the MTX-induced EMT in the alveolar epithelial cell line A549. MTX treatment decreased the mRNA expression of heme oxidase-1 (HO-1), a target of Nrf2, which was inhibited by co-treatment with diethyl maleate (DEM), an Nrf2 activator. Additionally, the MTX-induced increase in reactive oxygen species (ROS) production was significantly suppressed by DEM. Furthermore, DEM decreased mRNA/protein expression levels of α-smooth muscle actin (SMA), a representative EMT marker, which were upregulated by MTX. Nuclear expression and localization of Nrf2 were suppressed by MTX treatment, which led to a decrease in Nrf2 activity. Finally, in Nrf2 knockdown cells, the MTX-induced enhancement of α-SMA mRNA/protein expression was not observed, indicating that downregulation of Nrf2 may play a critical role in the MTX-induced EMT in A549 cells. These results suggest that Nrf2-regulated transcriptional activity would be associated with the MTX-induced EMT induction.

Effect of Corticosteroids on Peptide Transporter 2 Function and Induction of Innate Immune Response by Bacterial Peptides in Alveolar Epithelial Cells.

In the lung alveolar region, the innate immune system serves as an important host defense system. We recently reported that peptide transporter 2 (PEPT2) has an essential role in the uptake of bacterial peptides and induction of innate immune response in alveolar epithelial cells. In this study, we aimed to clarify the effects of corticosteroids on PEPT2 function and PEPT2-dependent innate immune response. NCI-H441 (H441) cells were used as an in vitro model of human alveolar type II epithelial cells, and the effects of dexamethasone (DEX) and budesonide (BUD) on the transport function of PEPT2 and the innate immune response induced by bacterial peptides were examined. PEPT2 function, estimated by measuring ß-alanyl-Nε-(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)-L-lysine (ß-Ala-Lys-AMCA) uptake in H441 cells, was suppressed by treatment with DEX and BUD in a concentration- and time-dependent manner. The suppression of PEPT2 function was partially recovered by a glucocorticoid receptor antagonist. The expression of PEPT2 and nucleotide-binding oligomerization domain 1 (NOD1) mRNAs was suppressed by treatment with DEX and BUD, while PEPT2 protein level was not changed by these treatment conditions. Additionally, the increased mRNA expression of interleukin (IL)-8 and the increased secretion of IL-8 into the culture medium induced by bacterial peptides were also suppressed by treatment with these corticosteroids. Taken together, these results clearly suggest that corticosteroids suppress PEPT2 function and bacterial peptide-induced innate immune response in alveolar epithelial cells. Therefore, PEPT2- and NOD1-dependent innate immune response induced by bacterial peptides in the lung alveolar region may be suppressed during the inhaled corticosteroid therapy.

Characterization of miR-34a-Induced Epithelial-Mesenchymal Transition in Non-Small Lung Cancer Cells Focusing on p53.

BACKGROUND: Epithelial-mesenchymal transition (EMT), a phenotypic conversion of the epithelial to mesenchymal state, contributes to cancer progression. Currently, several microRNAs (miRNAs) are associated with EMT-mediated cancer progression, but the contribution of miR-34a to EMT in cancer cells remains controversial. The present study aimed to clarify the role of miR-34a in the EMT-related phenotypes of human non-small cell lung cancer (NSCLC) cell lines, A549 (p53 wild-type) and H1299 (p53-deficient). METHODS: The miR-34a mimic and p53 small interfering RNA (siRNA) were transfected into the cells using Lipofectamine, and the obtained total RNA and cell lysates were used for real-time polymerase chain reaction and Western blotting analysis, respectively. RESULTS: The introduction of the miR-34a mimic led to an increase in the mRNA and protein expression levels of α-smooth muscle actin (α-SMA), a mesenchymal marker gene, in A549, but not in H1299 cells. Additionally, miR-34a-induced the upregulation of p53 activity and migration was observed in A549, but not in H1299 cells. However, under the p53-knockdown condition, only α-SMA upregulation by miR-34a was abolished. CONCLUSION: These findings indicate a close relationship between p53 and miR-34a-induced EMT in p53-wild type NSCLC cells, which provides novel insights about the role of miR-34a in EMT-like phenotypic changes in NSCLC.

Evaluation on epithelial-mesenchymal state and microRNAs focusing on isolated alveolar epithelial cells from bleomycin injured rat lung.

Several studies using bleomycin (BLM)-induced lung injury rat model revealed that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. Conversely, microRNAs (miRNAs) are considered as useful markers of various diseases. In the present study, we aimed to characterize the EMT state through focusing on alveolar epithelial cells and identify the miRNAs that can be used as markers to predict pulmonary fibrosis using a BLM-induced lung injury rat model. Intratracheal administration of BLM increased hydroxyproline, a component of collagen, in lung tissues at day 14, but not at day 7. However, BLM induced EMT at day 7, which was accompanied with increased mRNA expression of α-smooth muscle actin, a representative EMT marker, in alveolar epithelium, thereby suggesting that EMT occurs prior to pulmonary fibrosis in alveolar epithelial cells. Using this rat model, the expression levels of several EMT-associated miRNAs were examined, and miR-222 was found to be upregulated in alveolar epithelial cells as well as bronchoalveolar lavage fluid from day 3. Our findings indicate that EMT in alveolar epithelial cells may occur before pulmonary fibrosis, and miR-222 may be used as a potential marker for early prediction of pulmonary fibrosis.

Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells.

Epithelial-mesenchymal transition (EMT), a biological process through which epithelial cells transdifferentiate into mesenchymal cells, is involved in several pathological events, such as cancer progression and organ fibrosis. So far, we have found that methotrexate (MTX), an anticancer drug, induced EMT in the human A549 alveolar adenocarcinoma cell line. However, the relationship between EMT and the cytotoxicity induced by MTX remains unclear. In this study, we compared the processes of MTX-induced EMT and apoptosis in A549 cells. Q-VD-Oph, a caspase inhibitor, suppressed MTX-induced apoptosis, but not the increase in mRNA expression of α-smooth muscle actin (SMA), a representative EMT marker. In addition, SB431542, an EMT inhibitor, did not inhibit MTX-induced apoptosis. By using isolated clonal cells from wild-type A549 cells, the induction of EMT and apoptosis by MTX in each clone was analyzed, and no significant correlation was observed between the MTX-induced increase in α-SMA mRNA expression and the proportion of cells undergoing apoptosis. Furthermore, the increase in the mRNA expression of α-SMA was well correlated with cyclin-dependent kinase inhibitor 1A, a cell cycle arrest marker, but not with BCL-2 binding component 3 and Fas cell surface death receptor, which are both pro-apoptotic factors, indicating that the MTX-induced EMT may be related to cell cycle arrest, but not to apoptosis. These findings suggested that different mechanisms were involved in the MTX-induced EMT and apoptosis.

miR-484: A Possible Indicator of Drug-Induced Pulmonary Fibrosis.

BACKGROUND: Drug-induced lung injury leads to serious lung diseases, such as pulmonary fibrosis. We demonstrated in an alveolar epithelial cell line A549/ABCA3 that certain microRNAs were associated with bleomycin induced epithelial-mesenchymal transition (EMT) which is closely related to pulmonary fibrosis. In this study, we focused on the role of miR-484 in drug-induced EMT using A549/ABCA3 cells and a mouse lung injury model. METHODS: The expression of EMT-related genes and miR-484 was detected by real-time polymerase chain reaction. miR-484-targeted proteins were analyzed by Western blot. Pulmonary fibrosis mouse model was prepared by the intratracheal administration of BLM. As miR-484 is known to target SMAD2 and zinc finger E-box binding homeobox 1 (ZEB1), which are the well-known EMT-related transcription factors, we assessed the effects of a miR-484 inhibitor or mimic on the mRNA/protein expression of both the factors. RESULTS: We found that bleomycin significantly suppressed the intracellular expression and extracellular release of miR-484 in A549/ABCA3 cells. Moreover, the miR-484 mimic and inhibitor showed no drastic effects on the expression of the EMT-related transcription factors. In addition, the miR-484 mimic had no effect on the bleomycin-induced altered mRNA expression of the α-smooth muscle actin, a representative EMT marker. This suggested that miR-484 did not directly contribute to bleomycin-induced EMT in A549/ABCA3 cells. In contrast, the significant decrease in miR-484 expression in the lung tissue or plasma of bleomycin-administered mice suggested that miR-484 expression was closely correlated with bleomycin-induced lung injury. CONCLUSIONS: These findings indicate that miR-484 could be a novel diagnostic indicator for drug-induced pulmonary fibrosis.

Suppressive effect of quercetin against bleomycin-induced epithelial-mesenchymal transition in alveolar epithelial cells.

Quercetin is a flavonol that is known to have numerous beneficial biological effects such as an anti-fibrotic effect. Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells is one of major causes of pulmonary fibrosis. However, the effect of quercetin on drug-induced EMT in alveolar type II cells is not known. In this study, we examined the effect of quercetin on bleomycin (BLM)-induced EMT using RLE/Abca3 cells having alveolar type II cell-like phenotype. BLM induced EMT-like morphological changes, downregulation of an epithelial marker E-cadherin, and upregulation of a mesenchymal marker α-smooth muscle actin in RLE/Abca3 cells. In addition, BLM increased the levels of phosphorylated Smad2 and Slug mRNA expression, and enhanced nuclear translocation of ß-catenin, suggesting that BLM induced EMT in RLE/Abca3 cells via Smad and ß-catenin signaling pathways. However, when the cells were co-treated with quercetin, quercetin suppressed all of these EMT-related changes induced by BLM. Furthermore, BLM increased the intracellular level of reactive oxygen species, which was also suppressed by quercetin. These results suggest that quercetin may be a possible candidate for preventing pulmonary fibrosis caused by drugs.

Role of plasminogen activator inhibitor-1 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells.

There is increasing evidence that epithelial-mesenchymal transition (EMT) contributes to the development of organ fibrosis. We demonstrated that methotrexate (MTX) clearly induced EMT through the transforming growth factor (TGF)-ß-related signaling pathway in human alveolar epithelial cell line, A549. However, critical factors associated with MTX-induced EMT have not yet been identified. In our study, we attempted to identify factors playing a crucial role in MTX-induced EMT in A549 cells. We focused on plasminogen activator inhibitor-1 (PAI-1) as the possible target for the prevention of MTX-induced EMT-related lung injury. Comprehensive gene expression analysis by microarray revealed that mRNA expression level of PAI-1 was clearly increased by MTX treatment. In addition, using several cloned A549 cells, we found a good correlation between MTX-induced increase in mRNA expression levels of α-smooth muscle actin (SMA), a representative EMT marker, and PAI-1. Furthermore, MTX upregulated mRNA and protein expression levels of PAI-1 in A549 cells; this upregulation was canceled by co-treatment with SB431542, a TGF-ß-related signaling pathway inhibitor. Notably, tiplaxtinin, a PAI-1 inhibitor, and knockdown of urokinase-type plasminogen activator receptor (uPAR) prevented MTX-induced EMT in A549 cells. These findings indicate that MTX may induce EMT via upregulation of PAI-1 expression and interaction of PAI-1 with uPAR in A549 cells.

Suppression of P-glycoprotein by cigarette smoke extract in human lung-derived A549/P-gp cells.

Effect of long-term treatment with cigarette smoke extract (CSE) on the function and expression of P-glycoprotein (P-gp) in lung alveolar epithelial cells was examined using A549/P-gp cell line expressing P-gp. CSE treatment suppressed P-gp activity in a concentration- and treatment time-dependent manner. The suppression of P-gp activity by CSE was irreversible for at least 96 h after removal of CSE. In addition, CSE treatment suppressed the expression of P-gp mRNA and protein. In order to understand the mechanisms underlying P-gp suppression by CSE, the role of reactive oxygen species (ROS) was examined. CSE treatment increased intracellular ROS level, and suppressed catalase activity. α-Tocopherol suppressed ROS production by CSE, and ameliorated the suppression of P-gp activity by CSE, suggesting that ROS is involved in CSE-induced suppression of P-gp. The role of intracellular signaling pathways such as the nuclear factor κB and mitogen-activated protein kinase pathways was also examined. Among these pathways, the involvement of extracellular signal-regulated kinase (ERK) pathway was suggested. Taken together, long-term CSE treatment may suppress P-gp via modulation of ROS level and ERK pathway in alveolar epithelial cells.

Effect of transforming growth factor-ß1 on functional expression of monocarboxylate transporter 1 in alveolar epithelial A549 cells.

Epithelial-mesenchymal transition (EMT) contributes to the development of severe lung diseases, such as pulmonary fibrosis. Recently, it has been reported that EMT involves complex metabolic reprogramming triggered by several factors including transforming growth factor (TGF-ß1) and that monocarboxylate transporter (MCT1) plays an essential role in these metabolic changes. The aim of the present study was to clarify the functional expression of MCT1 during TGF-ß1-induced EMT in alveolar epithelial A549 cells. The transport function of MCT1 in A549 cells was examined using [3H]γ-hydroxybutyrate (GHB) and [3H] lactic acid (LA) as substrates and α-cyano-4-hydroxycinnamate (CHC), lactic acid, phloretin, and AR-C155858 (AR) as inhibitors of MCT1. EMT was induced by treating the cells with TGF-ß1. mRNA and protein expression levels were analyzed using real-time PCR and Western blotting, respectively. Time-, temperature-, and pH-dependent GHB and LA uptake were observed in A549 cells. CHC, lactic acid, phloretin, and AR significantly inhibited the uptake of GHB in a concentration-dependent manner, suggesting that MCT1 is primarily responsible for transport of monocarboxylates such as GHB and LA in A549 cells. TGF-ß1 treatment significantly enhanced GHB and LA uptake as well as the mRNA and protein expression levels of MCT1 in A549 cells. These changes were neutralized by co-treatment with SB431542, an inhibitor for the TGF-ß1 signaling pathway. CHC and AR had no effect on TGF-ß1-induced EMT-related gene expression changes. Here, we have clearly characterized functional expression of MCT1 in A549 cells and have shown that MCT1 may be upregulated via the TGF-ß1 signaling pathway.

Investigation on inhibitory effect of folic acid on methotrexate-induced epithelial-mesenchymal transition focusing on dihydrofolate reductase.

Use of methotrexate (MTX) can induce serious adverse lung reactions, such as pulmonary fibrosis. Recently, we demonstrated that the epithelial-mesenchymal transition (EMT), which triggers pulmonary fibrosis, was induced by MTX, and folic acid (FA) suppressed MTX-induced EMT in A549 cells. In this study, the role of dihydrofolate reductase (DHFR), a target of MTX, in FA-mediated inhibition of MTX-induced EMT was evaluated. The inhibitory effects of FA and tetrahydrofolate (THF), a metabolite of FA produced by DHFR, on MTX-induced increases in mRNA expression of α-SMA, an EMT marker, were compared. The IC50 values of FA and THF for DHFR were 103.3 and 19.4 µM, respectively. In contrast, DHFR knockdown did not alter the mRNA expression of α-SMA. Notably, the inhibitory effect of FA, but not THF, on MTX-induced EMT was blunted in DHFR knockdown cells. These results suggested that DHFR may not directly contribute to MTX-induced EMT, but may contribute to suppression of MTX-induced EMT via production of THF in A549 cells.

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells.

PURPOSE: Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. METHODS: Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. RESULTS: BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. CONCLUSIONS: These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells.

Association of cell cycle arrest with anticancer drug-induced epithelial-mesenchymal transition in alveolar epithelial cells.

Many drugs exert serious cytotoxic effects on pulmonary tissues. Although several reports have shown an association of epithelial-mesenchymal transition (EMT) with anticancer drug-induced lung injury, mechanisms of these effects are poorly understood. In the present study, we evaluated mechanisms of anticancer drug-induced EMT, with a focus on involvement of cell cycle arrest. We found that methotrexate (MTX) altered mRNA expression levels of many genes as determined by microarray analysis. Gene set enrichment analysis revealed that cell cycle arrest pathways may be associated with MTX-induced EMT. In addition, thymidine (THY) and nocodazole (NOC), which induce cell cycle arrest at S-phase and G2/M-phase, increased mRNA expression levels of α-smooth muscle actin (SMA), an EMT marker. Furthermore, α-SMA protein expression in cells arrested at S- and G2/M-phases by MTX and paclitaxel (PTX) was significantly higher than that in cells at G1. Notably, co-treatment of cells with THY or NOC and EMT-inducing anticancer drugs did not result in additional upregulation of α-SMA mRNA expression. These findings suggested that cell cycle arrest may be closely associated with anticancer drug-induced EMT in alveolar epithelial cells.

Role of peptide transporter 2 and MAPK signaling pathways in the innate immune response induced by bacterial peptides in alveolar epithelial cells.

AIMS: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549. MAIN METHODS: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium. KEY FINDINGS: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response. SIGNIFICANCE: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.

Reduced folate carrier-mediated methotrexate transport in human distal lung epithelial NCl-H441 cells.

OBJECTIVES: We had previously found that reduced folate carrier (RFC; SLC19A1) is mainly involved in an influx of transport of methotrexate (MTX), a folate analogue, using alveolar epithelial A549 cells. Therefore, we examined MTX uptake in NCl-H441 (H441) cells, another in vitro alveolar epithelial model, focusing on the localization of RFC in the present study. METHODS: Transport function of RFC in H441 cells was studied using [3 H]MTX. KEY FINDINGS: The uptake of MTX was increased remarkably after pretreatment of the cell monolayer with ethylenediaminetetraacetic acid (EDTA) in H441 cells but not in A549 cells, indicating the contribution of the basolaterally located transporter. In addition, folic acid and thiamine monophosphate, RFC inhibitors, inhibited the uptake of MTX from the basolateral side of the H441 cells. In order to compare the function of RFC on the apical and basolateral sides of the cells, the uptake of MTX from each side was examined using a Transwell chamber. Intracellular MTX amounts from the basolateral side were found to be significantly higher than those from the apical side. CONCLUSIONS: These findings suggest that the distribution of MTX in the lung alveolar epithelial cells may be mediated by basolaterally located RFC in alveolar epithelial cells.

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell.

PURPOSE: Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. METHODS: FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. RESULTS: FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. CONCLUSION: AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Folic acid prevents methotrexate-induced epithelial-mesenchymal transition via suppression of secreted factors from the human alveolar epithelial cell line A549.

Methotrexate (MTX) often induces serious lung diseases such as pulmonary fibrosis. Although MTX is known to be a folic acid (FA) antagonist, the effect of FA on MTX-induced lung injury remains unclear. Recent studies indicate that epithelial-mesenchymal transition (EMT) is involved in pulmonary fibrosis. Here, we aimed to clarify the effect of FA on MTX-induced EMT in human alveolar epithelial cell line A549 using conditioned medium (CM). CM was prepared from the supernatants of A549 cells treated with MTX in the absence (CMM) or presence (CMMF) of FA. FA suppressed EMT-like morphological changes and elevated mRNA/protein expression levels of α-smooth muscle actin induced by MTX in A549 cells. In addition, CMM induced EMT-like phenotypical changes, whereas CMMF had no effect on the phenotype of A549 cells, indicating that FA may suppress MTX-induced EMT via inhibiting the secretion of certain factors into the supernatant of the cells. Furthermore, FA also prevented CMM-induced EMT-like phenotypical changes in A549 cells. These findings indicate that FA may be a useful pharmaceutical for MTX-induced lung injury.

Nicotine transport in lung and non-lung epithelial cells.

AIMS: Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. MATERIALS AND METHODS: Characteristics of [3H]nicotine uptake was studied using these cell lines. KEY FINDINGS: Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. SIGNIFICANCE: The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed.

Effect of COA-Cl on transforming growth factor-ß1-induced epithelial-mesenchymal transition in RLE/Abca3 cells.

Transforming growth factor (TGF)-ß1 has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) in pathological conditions such as cancer and organ fibrosis. In this study, we examined the effect of a novel nucleic acid analog, COA-Cl, on TGF-ß1-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Changes in the cell morphology consistent with EMT were induced by TGF-ß1, whereas, this response was suppressed by co-treatment of the cells with COA-Cl. In addition, co-treatment with COA-Cl abolished TGF-ß1-induced downregulation of cytokeratin 19 and upregulation of α-smooth muscle actin transcripts. In order to delineate the mechanism underlying the inhibitory effect of COA-Cl on TGF-ß1-induced EMT in RLE/Abca3 cells, we examined the role of zinc finger E-box binding homeobox (ZEB) family transcription factors in this phenomenon. Our results demonstrated that the treatment of cells with COA-Cl suppressed the TGF-ß1 mediated increase in the mRNA levels of ZEB2. Overall, it was concluded that COA-Cl may have an inhibitory effect on TGF-ß1-induced EMT-like phenotypical changes in RLE/Abca3 cells via suppression of ZEB2 mRNA expression.