Masashi Kawami

Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar epithelial cells

Introduction: Pulmonary delivery is an attractive administration route for therapeutic proteins and peptides. In this context, endocytosis/transcytosis at the distal lung epithelial barrier is an important process in the pulmonary absorption of therapeutic macromolecules. The alveolar epithelium is comprised of type I and type II cells. Understanding the transport mechanisms in these cells is essential for the development of efficient pulmonary delivery systems of therapeutic macromolecules. Areas covered: Endocytic pathways for albumin and insulin in alveolar epithelial cells and possible receptors for the endocytosis are discussed. Strategies to enhance the endocytosis and pulmonary absorption of macromolecules are also discussed, by focusing on the effects of cationic poly(amino acid)s. Expert opinion: Although the surface area occupied by type II cells in alveoli is much smaller than that covered by type I cells, type II cells may significantly contribute to the endocytosis/transcytosis of macromolecules such as albumin. Identification of the receptors involved in the cellular uptake of each macromolecule is prerequisite for the understanding and regulation of its transport into and across alveolar epithelial cells. Establishment of novel in-vitro culture cell models of type I and type II cells would be a great help for the future advance of this research field.

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

PurposeMethotrexate (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.MethodsFirst, 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.ResultsAfter 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.ConclusionWe 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.

Analysis of TGF-β1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3.

In this study, we examined the induction of epithelial-mesenchymal transition (EMT) by transforming growth factor (TGF)-β1 and drugs in genetically engineered type II alveolar epithelial cell line RLE/Abca3. Treatment of RLE/Abca3 cells with TGF-β1 induced marked changes in cell morphology from epithelial-like to elongated fibroblast-like morphology. With these morphological changes, mRNA expression of epithelial markers such as cytokeratin 19 (CK19) decreased, while that of mesenchymal markers such as α-smooth muscle actin (α-SMA) increased. TGF-β1 treatment also decreased the mRNA expression of Abca3, a type II cell marker, and formation of lamellar body structures. Interestingly, the effect of TGF-β1 on Abca3 mRNA expression was observed in RLE/Abca3 cells, but not in wild-type RLE-6TN, A549, and H441 cells. Treatment of RLE/Abca3 cells with bleomycin (BLM) and methotrexate (MTX) induced similar morphological and mRNA expression changes. In addition, the increase in α-SMA and the decrease in Abca3 mRNA expression by these drugs were observed only in RLE/Abca3 cells. These findings suggest that, like TGF-β1, BLM and MTX induce EMT in RLE/Abca3 cells, and RLE/Abca3 cells would be a good model to study drug-induced EMT. The effect of pirfenidone, an antifibrotic and anti-inflammatory drug, on EMT induced by TGF-β1 was also discussed.

Role of miR-34a in TGF-β1- and Drug-Induced Epithelial-Mesenchymal Transition in Alveolar Type II Epithelial Cells

Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells may play an important role in the pulmonary fibrosis induced by drugs such as bleomycin (BLM) and methotrexate (MTX). In this study, we examined the role of microRNAs (miRNAs) in drug-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Based on the screening using miRNA microarray analysis, it was found that the expression of some miRNAs, such as miR-34a, was increased by transforming growth factor (TGF)-β1 and BLM. An increase in miR-34a expression due to TGF-β1, BLM, and MTX was also observed in real-time PCR analysis. Therefore, miR-34a was focused upon in further studies. The expression of nectin-1 mRNA and protein, a possible target of miR-34a, was decreased by the treatment with TGF-β1, BLM, and MTX. In addition, when RLE/Abca3 cells were transfected with miR-34a mimic, the expression of nectin-1 mRNA and Abca3 mRNA, another target of miR34a, decreased significantly. Furthermore, the mRNA expression of cytokeratin 19, an epithelial marker, decreased, whereas that of α-smooth muscle actin, a mesenchymal marker, increased in the cells transfected with miR-34a mimic. These results suggest that miR-34a is involved in drug-induced EMT in alveolar epithelial cells, and possibly in lung fibrosis.

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-β1-induced epithelial–mesenchymal transition in RLE/Abca3 cells

Transforming growth factor (TGF)-β1 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-β1-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-β1, whereas, this response was suppressed by co-treatment of the cells with COA-Cl. In addition, co-treatment with COA-Cl abolished TGF-β1-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-β1-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-β1 mediated increase in the mRNA levels of ZEB2. Overall, it was concluded that COA-Cl may have an inhibitory effect on TGF-β1-induced EMT-like phenotypical changes in RLE/Abca3 cells via suppression of ZEB2 mRNA expression.

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

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.

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.