Effect of transforming growth factor-β1 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-β1) 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-β1-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-β1. 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-β1 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-β1 signaling pathway. CHC and AR had no effect on TGF-β1-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-β1 signaling pathway.This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences (JSPS) (numbers; 26293033, 15 K08074, and 16 K18945)

Enhancement effect of poly(amino acid)s on insulin uptake in alveolar epithelial cells.

In this study, we elucidated the effect of poly(amino acid)s such as poly-L-ornithine (PLO) on FITC-insulin uptake in cultured alveolar type II epithelial cells, RLE-6TN. FITC-insulin uptake by RLE-6TN cells as well as its cell surface binding was markedly increased by PLO without cytotoxicity. The uptake of FITC-insulin in the presence of PLO was shown to be mediated by endocytosis, but in contrast to the uptake in the absence of PLO, the contribution of macropinocytosis emerged. Colocalization of FITC-insulin and LysoTracker Red was observed by confocal laser scanning microscopy both in the absence and presence of PLO, indicating that FITC-insulin was partly targeted to lysosomes in the cells and degraded. The half-life of the intracellular degradation of FITC-insulin was, however, prolonged by the presence of PLO. PLO also stimulated the uptake of other FITC-labeled compounds. Among them, the enhancement effects of PLO on FITC-albumin and FITC-insulin uptake were prominent. The effect of PLO on insulin absorption was also examined in in-vivo pulmonary administration in rats, and co-administration of PLO enhanced the hypoglycemic action of insulin. These findings suggest that co-administration of poly(amino acid)s such as PLO is a useful strategy for enhancing insulin uptake by alveolar epithelial cells and subsequent absorption from the lung.In this study, we elucidated the effect of poly(amino acid)s such as poly-L-ornithine (PLO) on FITC-insulin uptake in cultured alveolar type II epithelial cells, RLE-6TN. FITC-insulin uptake by RLE-6TN cells as well as its cell surface binding was markedly increased by PLO without cytotoxicity. The uptake of FITC-insulin in the presence of PLO was shown to be mediated by endocytosis, but in contrast to the uptake in the absence of PLO, the contribution of macropinocytosis emerged. Colocalization of FITC-insulin and LysoTracker Red was observed by confocal laser scanning microscopy both in the absence and presence of PLO, indicating that FITC-insulin was partly targeted to lysosomes in the cells and degraded. The half-life of the intracellular degradation of FITC-insulin was, however, prolonged by the presence of PLO. PLO also stimulated the uptake of other FITC-labeled compounds. Among them, the enhancement effects of PLO on FITC-albumin and FITC-insulin uptake were prominent. The effect of PLO on insulin absorption was also examined in in-vivo pulmonary administration in rats, and co-administration of PLO enhanced the hypoglycemic action of insulin. These findings suggest that co-administration of poly(amino acid)s such as PLO is a useful strategy for enhancing insulin uptake by alveolar epithelial cells and subsequent absorption from the lung

Gadolinium modulates gentamicin uptake via an endocytosis-independent pathway in HK-2 human renal proximal tubular cell line

The aim of this study was to characterize the uptake mechanism of gentamicin, an aminoglycoside antibiotic, in human renal proximal tubular cell line HK-2. Sodium-dependent uptake of D-[H-3]glucose and L-[H-3]alanine was observed in HK-2 cells, indicating that the cells employed in this study retain functional characteristics of the renal proximal tubular cells. On the other hand, mRNA and protein expression of megalin, an endocytic receptor which is responsible for the internalization of gentamicin into the renal proximal tubular cells, was very faint in HK-2 cells. Various aminoglycoside antibiotics including amikacin and kanamycin inhibited the uptake of [H-3]gentamicin. Colchicine and cytochalasin D, general endocytosis inhibitors, had no significant effect on [H-3]gentamicin uptake in HK-2 cells, which was in contrast to the results observed in OK cells, a renal proximal tubular cell line expressing megalin. Furthermore, unlike OK cells, [H-3]gentamicin uptake in HK-2 cells was not inhibited by N-WASP181-200, a cationic 20-amino acid peptide. Ruthenium red, a nonspecific cation channel blocker, decreased the uptake of [H-3]gentamicin in HK-2 cells. In contrast, the trivalent cation gadolinium biphasically modulated [H-3]gentamicin uptake with a maximum increase at 0.3 mM gadolinium. The enhanced effect of gadolinium on [H-3]gentamicin uptake was independent of gadolinium-induced increase in intracellular calcium concentration. These findings indicate that gentamicin is primarily taken up via an endocytosis-independent pathway in HK-2 cells with very low expression of megalin, and that the uptake of gentamicin is modulated by gadolinium

Mechanism underlying insulin uptake in alveolar epithelial cell line RLE-6TN

For the development of efficient pulmonary delivery systems for protein and peptide drugs, it is important to understand their transport mechanisms in alveolar epithelial cells. In this study, the uptake mechanism for FITC-insulin in cultured alveolar epithelial cell line RLE-6TN was elucidated. FITC-insulin uptake by RLE-6TN cells was time-dependent, temperature-sensitive, and concentration-dependent. The uptake was inhibited by metabolic inhibitors, cytochalasin D, clathrin-mediated endocytosis inhibitors, and dynasore, an inhibitor of dynamin GTPase. On the other hand, no inhibitory effect was observed with caveolae-mediated endocytosis inhibitors and a macropinocytosis inhibitor. Intracellular FITC-insulin was found to be partly transported to the basal side of the epithelial cell monolayers. In addition, colocalization of FITC-insulin and LysoTracker Red was observed on confocal laser scanning microscopy, indicating that FITC-insulin was partly targeted to lysosomes. In accordance with these findings, SDS-PAGE/fluoroimage analysis showed that intact FITC-insulin in the cells was eliminated with time. The possible receptor involved in FITC-insulin uptake by RLE-6TN cells was examined by using siRNA. Transfection of the cells with megalin or insulin receptor siRNA successfully reduced the corresponding mRNA expression. FITC-insulin uptake decreased on the transfection with insulin receptor siRNA, but not that with megalin siRNA. These results suggest that insulin is taken up through endocytosis in RLE-6TN cells, and after the endocytosis, the intracellular insulin is partly degraded in lysosomes and partly transported to the basal side. Insulin receptor, but not megalin, may be involved at least partly in insulin endocytosis in RLE-6TN cells

Mechanism underlying insulin uptake in alveolar epithelial cell line RLE-6TN.

For the development of efficient pulmonary delivery systems for protein and peptide drugs, it is important to understand their transport mechanisms in alveolar epithelial cells. In this study, the uptake mechanism for FITC-insulin in cultured alveolar epithelial cell line RLE-6TN was elucidated. FITC-insulin uptake by RLE-6TN cells was time-dependent, temperature-sensitive, and concentration-dependent. The uptake was inhibited by metabolic inhibitors, cytochalasin D, clathrin-mediated endocytosis inhibitors, and dynasore, an inhibitor of dynamin GTPase. On the other hand, no inhibitory effect was observed with caveolae-mediated endocytosis inhibitors and a macropinocytosis inhibitor. Intracellular FITC-insulin was found to be partly transported to the basal side of the epithelial cell monolayers. In addition, colocalization of FITC-insulin and LysoTracker Red was observed on confocal laser scanning microscopy, indicating that FITC-insulin was partly targeted to lysosomes. In accordance with these findings, SDS-PAGE/fluoroimage analysis showed that intact FITC-insulin in the cells was eliminated with time. The possible receptor involved in FITC-insulin uptake by RLE-6TN cells was examined by using siRNA. Transfection of the cells with megalin or insulin receptor siRNA successfully reduced the corresponding mRNA expression. FITC-insulin uptake decreased on the transfection with insulin receptor siRNA, but not that with megalin siRNA. These results suggest that insulin is taken up through endocytosis in RLE-6TN cells, and after the endocytosis, the intracellular insulin is partly degraded in lysosomes and partly transported to the basal side. Insulin receptor, but not megalin, may be involved at least partly in insulin endocytosis in RLE-6TN cells.For the development of efficient pulmonary delivery systems for protein and peptide drugs, it is important to understand their transport mechanisms in alveolar epithelial cells. In this study, the uptake mechanism for FITC-insulin in cultured alveolar epithelial cell line RLE-6TN was elucidated. FITC-insulin uptake by RLE-6TN cells was time-dependent, temperature-sensitive, and concentration-dependent. The uptake was inhibited by metabolic inhibitors, cytochalasin D, clathrin-mediated endocytosis inhibitors, and dynasore, an inhibitor of dynamin GTPase. On the other hand, no inhibitory effect was observed with caveolae-mediated endocytosis inhibitors and a macropinocytosis inhibitor. Intracellular FITC-insulin was found to be partly transported to the basal side of the epithelial cell monolayers. In addition, colocalization of FITC-insulin and LysoTracker Red was observed on confocal laser scanning microscopy, indicating that FITC-insulin was partly targeted to lysosomes. In accordance with these findings, SDS-PAGE/fluoroimage analysis showed that intact FITC-insulin in the cells was eliminated with time. The possible receptor involved in FITC-insulin uptake by RLE-6TN cells was examined by using siRNA. Transfection of the cells with megalin or insulin receptor siRNA successfully reduced the corresponding mRNA expression. FITC-insulin uptake decreased on the transfection with insulin receptor siRNA, but not that with megalin siRNA. These results suggest that insulin is taken up through endocytosis in RLE-6TN cells, and after the endocytosis, the intracellular insulin is partly degraded in lysosomes and partly transported to the basal side. Insulin receptor, but not megalin, may be involved at least partly in insulin endocytosis in RLE-6TN cells

Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells

Aims: The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine. employing human intestinal epithelial cell line Caco-2 as an in vitro model system. Main methods: Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. Key findings: Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A, and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0. Significance: The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis. which could occur by a process other than clathrin- and caveolin-dependent mechanisms

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