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Am J Physiol Lung Cell Mol Physiol 290: L946-L955, 2006. First published December 16, 2005; doi:10.1152/ajplung.00173.2005
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Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN

Ryoko Yumoto,1 Hiromi Nishikawa,1 Miho Okamoto,1 Hirokazu Katayama,2 Junya Nagai,1 and Mikihisa Takano1

1Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima; and 2Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Japan

Submitted 18 April 2005 ; accepted in final form 14 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITC-albumin by the cells was time and temperature dependent and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-beta-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed.

albumin clearance; caveolae-mediated endocytosis; endocytic receptor; modified albumin; protein transport

THE DISTAL AIR SPACES OF THE lung (alveolar region) are lined with a continuous epithelium comprising two major types of epithelial cells, type I and type II. Type I epithelial cells have a squamous morphology and cover ~90–95% of the alveolar surface area. Type II cells are cuboidal epithelial cells and cover ~5–10% of the surface area, although the number of type II cells in alveolar epithelia is similar to or more than that of type I cells. Type II cells also serve as progenitors of type I cells (13, 40). Alveolar lining fluid contains various physiologically important proteins such as albumin, immunoglobulin G, and transferrin. The concentration of albumin in alveolar fluid is usually much lower than that in the blood, whereas the concentration would increase to 40–65% of plasma level in hydrostatic pulmonary edema and to 75–95% in lung injury pulmonary edema. The clearance of serum proteins from the alveolar space is a critical process in recovery from pulmonary edema and in maintaining the normal alveolar milieu (17, 22). Therefore, understanding the mechanisms of protein transport in alveolar epithelial cells is important for the development of better therapeutic approaches. In addition, such information could provide new insights into improved strategies for pulmonary delivery of exogenous protein drugs into the systemic circulation.

Clearance mechanisms of serum proteins from the alveolar space have been studied for years; among the mechanisms proposed, endocytosis (transcytosis) in alveolar epithelial cells is probably most important (17, 22). However, detailed mechanisms underlying the protein transport in alveolar epithelial cells are still unclear. The established cell line is a powerful tool for studies of transport events at the cellular level. Recently, Driscoll et al. (11) established two immortalized alveolar epithelial cell lines from rats. One of them, RLE-6TN, is probably derived from a spontaneous transformant and contains lipid-containing inclusion bodies, such as alveolar type II epithelial cells (11). RLE-6TN cells have several characteristics that are similar to alveolar type II epithelial cells, including the expression of cytokeratin 19 (11, 32). So far, however, there is little information concerning the transport and/or endocytic functions of RLE-6TN cells.

In this study, we attempted to elucidate the protein transport system in RLE-6TN cells to evaluate the usefulness of this cultured cell line as an in vitro model system of alveolar epithelial cells and to obtain further information concerning the protein transport mechanisms in alveolar epithelial cells. For this purpose, FITC-albumin was used as a substrate, and general characteristics of FITC-albumin uptake by RLE-6TN cells, the fate of FITC-albumin taken up by the cells, and the role of caveolae- and clathrin-mediated endocytosis in the uptake were examined. The possible involvement of such endocytic receptors as megalin and cubilin and the physiological/pathophysiological roles of the albumin endocytic system are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. DMEM-nutrient mixture F-12 (Ham) (1:1) (DMEM/F-12), trypsin-EDTA, and penicillin-streptomycin were purchased from Invitrogen (Grand Island, NY). FBS was purchased from Daiichi Pure Chemicals (Tokyo, Japan). FITC-labeled BSA (FITC-albumin), FITC-inulin, FITC-dextran (FD-20S, average molecular size of 19.5 kDa), phenylarsine oxide, bafilomycin A1, indomethacin, methyl-beta-cyclodextrin, and nystatin were purchased from Sigma-Aldrich (St. Louis, MO). Chlorpromazine, 2,4-dinitrophenol, and formaldehyde solution were purchased from Nacalai Tesque (Kyoto, Japan). 2-Deoxy-D-glucose and maleic anhydride were purchased from Kanto Chemical (Tokyo, Japan), and sodium azide was from Katayama Chemical (Tokyo, Japan). Nucleic acid purification kit (Mag Extractor-RNA) and RT-PCR kit (Rever Tra Dash) were purchased from TOYOBO (Osaka, Japan). LysoTracker red (LysoTracker red DND-99) was purchased from Molecular Probes (Eugene, OR). All other chemicals used for the experiments were of the highest purity commercially available.

Cell culture. RLE-6TN cells were obtained from American Type Culture Collection (ATCC no. CRL-2300; Manassas, VA). RLE-6TN cells were cultured in DMEM/F-12 containing 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin, in an atmosphere of 5% CO2-95% air at 37°C, and subcultured every 6–7 days using 0.25% trypsin and 1 mM EDTA. RLE-6TN cells were used between passages 38 and 61. Fresh medium was replaced every 2 or 3 days, and the cells were used for the experiments on the fifth to seventh day after seeding.

Alkaline phosphatase activity. RLE-6TN cells were grown on 35-mm culture dishes for 6 days. The culture medium was aspirated, and the dishes were rinsed rapidly twice with 2 ml of ice-cold saline. The cells were scraped with a rubber policeman into 0.5 ml of ice-cold saline, and the dishes were rinsed again with 0.5 ml of ice-cold saline to improve the recovery of the cells. The cells were washed by centrifugation at 4°C for 3 min at 8,720 g. The pellet was resuspended in 2 ml of saline and homogenized with a hand homogenizer (Ultra-turrax T-8; IKA-Werke, Staufen, Germany). After centrifugation at 4°C for 20 min at 9,000 g, the supernatant (S9 fraction) was used for the determination of alkaline phosphatase activity. Briefly, 0.1 ml of the supernatant was added to 0.5 ml of glycine buffer (50 mM glycine, 5 mM MgCl2, and 1 mM ZnCl2, pH 9.2) containing 18 mM p-nitrophenyl phosphate. After incubation for 15–45 min at 37°C, the reaction was stopped with 2.5 ml of 0.6 M NaOH. After centrifugation at 1,610 g for 10 min, the absorbance of supernatant was read at 410 nm with the use of a spectrophotometer.

Analysis of mRNA expression of cytokeratin 19, caveolin-1, megalin, and cubilin. The mRNA expressions of cytokeratin 19, caveolin-1{alpha}, and two endocytic receptors, megalin and cubilin, were analyzed as described previously (38). Total RNA was isolated from RLE-6TN cells (passages 43 and 61 for cytokeratin 19 and cubilin, passage 46 for caveolin-1{alpha}, and passage 38 for megalin) and from rat lung and renal cortex with Mag Extractor-RNA. The total RNA (0.2 µg) was used for RT to generate cDNA using Rever Tra Dash, and the generated RT cDNA was used for the PCR amplification with a Program Temp control system PC-707 (ASTEC, Fukuoka, Japan). The primers for cytokeratin 19 were sense, 5'-GGTGGAAGTTTTAGTGGGGC-3'; and antisense, 5'-CGTAGTGTGGACAGCGACCT-3' (expected size of PCR product, 677 bp). The conditions for PCR were denaturation, 94°C for 1 min; annealing, 66°C for 1 min; and extension, 72°C for 30 s (29 cycles). The primers for caveolin-1{alpha} were sense, 5'-GGACATCTCTACACTGTTCC-3'; and antisense, 5'-AGGATGGCAAAGTAGATGC-3' (expected size of PCR product, 335 bp). The conditions for PCR were denaturation, 98°C for 10 s; annealing, 60°C for 20 s; and extension, 72°C for 15 s (36 cycles). The primers for megalin were sense, 5' -ACACCGCTTCTGCCGTCT-3'; and antisense, 5' -TCTGAGCACTCCCGAGGAAC-3' (expected size of PCR product, 636 bp). The conditions for PCR were denaturation, 94°C for 1 min; annealing, 68°C for 1 min; and extension, 72°C for 30 s (39 cycles). The primers for cubilin were sense, 5'-GCCTGCCCCATTTATCTCTTC-3'; and antisense, 5' -CGCCGTTTCTTACCTCCAA-3' (expected size of PCR product, 652 bp). The conditions for PCR were denaturation, 94°C for 1 min; annealing, 69°C for 1 min; and extension, 72°C for 30 s (35 cycles). The PCR products with or without reverse transcription were separated by electrophoresis through 2.0% agarose gels and visualized under ultraviolet light with ethidium bromide.

Uptake of FITC-labeled compound by RLE-6TN cells. Uptake experiments were performed as described previously (39). RLE-6TN cells grown on 35-mm culture dishes were used. After removal of the culture medium, each dish was washed and preincubated with PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 0.1 mM CaCl2, and 0.5 mM MgCl2, pH 7.4) (PBS buffer) supplemented with 5 mM D-glucose (PBS-G buffer) at 37°C for 10 min. Then, PBS-G buffer containing FITC-albumin (20 µg/ml), FITC-inulin (500 µg/ml), or FITC-dextran (500 µg/ml) was added to each dish, and the cells were incubated at 37°C or 4°C for a specified period.

For inhibition studies, RLE-6TN cells were preincubated with PBS or PBS-G buffer at 37°C in the absence or presence of the inhibitor as follows: 1 mM 2,4-dinitrophenol for 10 min in PBS buffer, 10 mM sodium azide plus 5 mM 2-deoxy-D-glucose for 10 min in PBS buffer, 100 nM bafilomycin A1 for 30 min in PBS-G buffer containing 0.1% DMSO, nystatin (5.4–54.0 µM) for 10 min in PBS-G buffer containing 0.5% DMSO, indomethacin (75–300 µM) for 10 min in PBS-G buffer, methyl-beta-cyclodextrin (2.5–15 mM) for 10 min in PBS-G buffer, phenylarsine oxide (1–30 µM) for 10 min in PBS-G buffer containing 0.5% DMSO, and chlorpromazine (8.4–140.7 µM) for 10 min in PBS-G buffer. The same vehicles were used for each control experiment. The cells were then incubated with 2 ml of the vehicle containing FITC-labeled compound in the absence or presence of the inhibitor at 37°C for 60 min. Bafilomycin A1 and phenylarsine oxide were used only in the preincubation buffer and were not added to the uptake buffer. To examine the effect of various types of albumin (see below) and EGTA, cells were preincubated with PBS-G buffer at 37°C for 10 min, and then 2 ml of PBS-G buffer containing FITC-albumin (20 µg/ml) with or without various types of albumin or 1 mM EGTA was added to each dish and the cells were incubated at 37°C for 60 min. The effects of potassium depletion and hypertonicity on FITC-albumin uptake in RLE-6TN cells were examined as reported by Hansen et al. (16). The buffer used for potassium depletion contained 140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and 1 mg/ml D-glucose (pH 7.4) (HEPES buffer). Control cells were incubated with the same buffer supplemented with 10 mM KCl. HEPES buffer supplemented with 450 mM sucrose and 10 mM KCl was used as hypertonic medium. Uptake of FITC-labeled compound in these treated or control RLE-6TN cells was examined as described above.

At the end of the incubation, the uptake buffer was aspirated, and the dishes were rinsed rapidly three times with 2 ml of ice-cold PBS buffer. The cells were scraped with a rubber policeman into 0.75 ml of ice-cold PBS buffer, and the dishes were rinsed again with 0.5 ml of ice-cold PBS buffer to improve the recovery of the cells. The cells were washed by centrifugation at 4°C for 3 min at 9,838 g twice. After the supernatant was aspirated, the pellet was solubilized in 1.2 ml of 0.1% Triton X-100 in PBS buffer without CaCl2 and MgCl2 at room temperature for 30 min and centrifuged for 3 min at 5,600 g. The supernatant was used for fluorescence and protein assays.

Preparation of chemically modified albumin and circular dichroism measurement. Formaldehyde-treated albumin (formylated-albumin) was prepared as reported by Horiuchi et al. (19). Briefly, 400 mg of BSA was dissolved in 5.2 ml of 0.45 M sodium carbonate buffer (pH 10.0), followed by centrifugation at 1,670 g for 10 min to remove insoluble debris. To the supernatant, 5.4 ml of formaldehyde solution (37% wt/vol) was added under stirring. After incubation at 37°C for 60 min, the solution was dialyzed overnight against 0.15 M NaCl and then against distilled water at 4°C. After centrifugation at 20,000 g for 30 min at 4°C, the supernatant was stored at –80°C before use. Maleic anhydride-treated albumin (maleylated-albumin) was prepared as reported previously (3, 5) with some modifications. Briefly, 400 mg of BSA was dissolved in 9.0 ml of 0.1 M sodium pyrophosphate buffer (pH 9.0), followed by centrifugation at 1,670 g for 10 min to remove insoluble debris. To the supernatant, 1.0 ml of 1.0 M maleic anhydride dissolved in 1,4-dioxane was added under stirring. After incubation for 5 min on ice, the solution was dialyzed overnight against 0.15 M NaCl and then against distilled water at 4°C. After centrifugation at 20,000 g for 30 min at 4°C, the supernatant was stored at –80°C before use. Circular dichroism measurements of native and chemically modified albumins and calculation of {alpha}-helical contents in these proteins were performed according to the method reported by Bito et al. (4). All measurements were performed at room temperature with a spectropolarimeter (J-720; Jasco, Tokyo, Japan), and far-UV circular dichroism spectra (200–250 nm) were obtained at a protein concentration of 0.36 µM.

Confocal laser scanning microscopy. RLE-6TN cells were grown on 35-mm glass bottom culture dishes for 5 days. The cells were incubated with FITC-albumin (20 µg/ml) for 60 min at 37°C or at 4°C as described above; after the cells were washed with ice-cold PBS buffer three times for 5 min each, fluorescence in the cells was observed by confocal laser scanning microscopy (LSM5 Pascal, Carl ZEISS). In some cases, 75 nM of LysoTracker red, a fluorescent lysosomal marker, was added to the uptake medium at 30 min after the initiation of FITC-albumin uptake, and the cells were incubated further for 30 min with LysoTracker red and FITC-albumin.

Evaluation of intactness of FITC-albumin in uptake buffer and in RLE-6TN cells. The intactness of FITC-albumin in the uptake buffer (20 µg/ml) before and after incubation with RLE-6TN cells for 30 or 60 min and in the cells after uptake for 10, 30, or 60 min was evaluated after separation by SDS-PAGE. The samples were solubilized in a loading buffer consisting of 2% SDS, 50 mM Tris·HCl, 10% glycerol, and 6% 2-mercaptoethanol. Then, the sample was subjected to SDS-PAGE with 10% polyacrylamide gel. After SDS-PAGE, the fluorescence intensity of the gel was analyzed by fluoroimage analyzer FLA-2000 (Fuji Photo Film, Tokyo, Japan), and the image was obtained with the aid of a Macintosh personal computer and software provided with the analyzer.

Other analytical methods. The amount of FITC-albumin, FITC-inulin, or FITC-dextran taken up by RLE-6TN cells was measured using a Hitachi fluorescence spectrophotometer F-3000 (Tokyo, Japan) at an excitation wavelength of 500 nm and an emission wavelength of 520 nm. Protein was determined by the Lowry method with BSA as the standard.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed by Student's t-test or by one-way ANOVA followed by the Tukey's test for multiple comparisons. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of cultured RLE-6TN cells. As a first step, we examined whether RLE-6TN cells employed in this study possessed similar characteristics with those of alveolar type II cells. Figure 1A shows the time-dependent metabolism of p-nitrophenyl phosphate by S9 fraction of RLE-6TN cells, indicating that RLE-6TN cells have alkaline phosphatase activity. The expression of cytokeratin 19 mRNA was examined by RT-PCR analysis using specific primer (Fig. 1B). The PCR products of the expected size (677 bp) were observed in samples of RLE-6TN cells at passages 43 and 61, as well as that of the lung, after reverse transcription. On the other hand, no band was detected when total RNA was subjected to PCR without reverse transcription, showing that the PCR products are specifically derived from cytokeratin 19 mRNA. Thus RLE-6TN cells were shown to have some characteristic features of alveolar type II epithelial cells, such as alkaline phosphatase activity and the expression of cytokeratin 19 mRNA.


Figure 1
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  		Fig. 1. Alkaline phosphatase activity (A) and expression of cytokeratin 19 mRNA (B) in RLE-6TN cells. A: metabolism of p-nitrophenyl phosphate by S9 fraction of RLE-6TN cells. Generation of p-nitrophenol with time was measured by a spectrophotometer at a wavelength of 410 nm. Each point represents the mean ± SE of 3 determinations. B: expressions of cytokeratin 19 mRNA in RLE-6TN cells (passages 43 and 61) and in rat lung were analyzed by RT-PCR. PCR products with [RT(+)] or without [RT(–)] reverse transcription were separated by electrophoresis through 2.0% agarose gels and visualized under ultraviolet light with ethidium bromide.

 
General characteristics of albumin uptake by RLE-6TN cells. Figure 2 shows the time course of FITC-albumin uptake by RLE-6TN cells. FITC-albumin was taken up by the cells at 37°C, and the uptake increased linearly for up to 60 min. In contrast, the uptake was almost completely suppressed, and time-dependent uptake was not observed at 4°C.


Figure 2
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  		Fig. 2. Time and temperature dependence of FITC-albumin uptake by RLE-6TN cells. Uptake of FITC-albumin (20 µg/ml) by confluent monolayers of RLE-6TN cells was measured at 37°C ({circ}) or 4°C (bullet). Each point represents the mean ± SE of 3 monolayers. *P < 0.05, significantly different from the value at 37°C.

 
Figure 3 shows the concentration dependence of FITC-albumin uptake by RLE-6TN cells. The uptake was saturable (Fig. 3A), and, as evidenced by the Eadie-Hofstee plot (Fig. 3B), the uptake was mediated by high- and low-affinity transport systems. The Km and maximum uptake rate (Vmax) for high-affinity system were calculated to be 0.13 mg/ml (1.9 µM) and 6.5 µg·mg protein–1·60 min–1, and those for low-affinity system were 8.7 mg/ml (130 µM) and 46.9 µg·mg protein–1·60 min–1, respectively.


Figure 3
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  		Fig. 3. Concentration dependence of FITC-albumin uptake by RLE-6TN cells. A: FITC-albumin uptake for 60 min at 37°C was measured in a concentration range between 20 µg/ml and 10 mg/ml. B: Eadie-Hofstee plot of the data. Each point represents the mean ± SE of 3 monolayers.

 
The effect of various concentrations of native (unlabeled) albumin on FITC-albumin (20 µg/ml) uptake by RLE-6TN cells was examined. At this concentration of FITC-albumin, the uptake is mediated predominantly by a high-affinity transport system (~90% of total uptake). As shown in Fig. 4A, the uptake of FITC-albumin was inhibited by the presence of native albumin in a concentration-dependent manner.


Figure 4
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  		Fig. 4. Effect of native (unlabeled) and chemically modified albumins on FITC-albumin uptake by RLE-6TN cells. A: uptake of FITC-albumin (20 µg/ml) by RLE-6TN cells was measured at 37°C in the absence ({circ}) or presence (bullet) of various concentrations of native albumin. Each point represents the mean ± SE of 3 monolayers. *P < 0.05, significantly different from control ({circ}). B: uptake of FITC-albumin (20 µg/ml) by RLE-6TN cells was measured at 37°C in the absence ({circ}) or presence of various concentrations of native (bullet), formylated ({blacktriangleup}), or maleylated ({blacksquare}) albumin. Each point represents the mean ± SE of 3 monolayers.

 
To examine the effect of chemically modified albumins on FITC-albumin uptake by RLE-6TN cells, formylated- and maleylated-albumin were prepared. Before the inhibition studies, the {alpha}-helical content in each protein was measured because {alpha}-helical content in chemically modified albumins is reportedly less than that in native albumin (4). As expected, the {alpha}-helical content in native, FITC-, formylated-, and maleylated-albumin was 51.6, 41.3, 32.4, and 31.8%, respectively, indicating that albumin was modified by formaldehyde and maleic anhydride. The inhibitory effects of these chemically modified albumins on FITC-albumin uptake were examined and compared with that of native albumin. As shown in Fig. 4B, both formylated- and maleylated-albumin inhibited FITC-albumin uptake by RLE-6TN cells in a concentration-dependent manner, and the inhibitory potencies of these chemically modified albumins were similar to that of native albumin.

To further elucidate the albumin uptake by RLE-6TN cells, effects of metabolic inhibitors were examined. Pretreatment of the cells with either 2,4-dinitrophenol or sodium azide plus 2-deoxy-D-glucose significantly inhibited FITC-albumin uptake (Fig. 5). In addition, FITC-albumin uptake was inhibited by pretreatment of the cells with bafilomycin A1, an inhibitor of vacuolar H+-ATPase (Fig. 5). These results indicate that albumin uptake by RLE-6TN cells is mediated by receptor-mediated endocytosis.


Figure 5
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  		Fig. 5. Effect of metabolic inhibitors (left) and an inhibitor of vacuolar H+-ATPase (right) on FITC-albumin uptake by RLE-6TN cells. Cells were treated with 2,4-dinitrophenol (DNP), sodium azide (NaN3) plus 2-deoxy-D-glucose (2DOG), or bafilomycin A1 (BAF) as described in MATERIALS AND METHODS, and the uptake of FITC-albumin (20 µg/ml) for 60 min was measured at 37°C. Each column represents the mean ± SE of 3 monolayers. *P < 0.05, significantly different from each control.

 
Fate of FITC-albumin taken up by RLE-6TN cells. RLE-6TN cells formed epithelial-like monolayers when plated on culture dishes (Fig. 6A). Intracellular localization of FITC-albumin was examined by confocal laser scanning microscopy. When RLE-6TN cells were incubated with FITC-albumin at 37°C for 60 min, punctate localization of fluorescence was observed in the cells (Fig. 6B). This phenomenon was not observed when incubation was at 4°C (Fig. 6C). When cells were incubated with FITC-albumin and LysoTracker red, a lysosomal marker, colocalization of these two sources of fluorescence was observed (Fig. 6, D–F), indicating that the part of FITC-albumin taken up by the cells was targeted to lysosomes. Some FITC-albumin in the cells, however, was not colocalized with LysoTracker red (Fig. 6, G–I) even after 60 min of incubation.


Figure 6
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  		Fig. 6. Phase-contrast (A) and confocal laser scanning (B–I) micrographs of RLE-6TN cells. A: RLE-6TN cells were grown on glass coverslips in 24-well culture plates for 6 days and then observed by phase-contrast microscopy (Axiovert 200M, Carl Zeiss). B and C: after FITC-albumin (20 µg/ml) uptake by RLE-6TN cells grown on glass bottom culture dishes for 60 min at 37°C (B) or at 4°C (C), cells were observed by confocal laser scanning microscopy. D–F: after incubation with FITC-albumin (20 µg/ml) for 60 min and LysoTracker red (75 nM) for 30 min at 37°C, cells were observed by confocal laser scanning microscopy [D: FITC-albumin (green), E: LysoTracker red (red), F: merged]. Colocalization of fluorescence derived from FITC-albumin and LysoTracker red is shown as yellow color in F. G–I: micrographs of the different field of the same experiments with D–F [G: FITC-albumin (green), H: LysoTracker red (red), I: merged].

 
The intactness of FITC-albumin in the cells was evaluated by fluoroimage analyzer after SDS-PAGE. As shown in Fig. 7A, FITC-albumin in the cells gradually degraded over time (~16% at 60 min), although the rest remained intact. This degradation occurred in the cells, probably in lysosomes, because FITC-albumin in the uptake buffer was highly stable, and degradation was negligible even after 60 min of incubation (Fig. 7B).


Figure 7
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  		Fig. 7. Estimation of intact FITC-albumin in RLE-6TN cells (A) and in uptake buffer (B). The intactness of FITC-albumin was evaluated by fluoroimage analyzer after separation by SDS-PAGE. A: cell samples after FITC-albumin (20 µg/ml) uptake for 10, 30, or 60 min. B: uptake buffer before (0 min) and after incubation with RLE-6TN cells for 30 or 60 min. The initial concentration of FITC-albumin was 20 µg/ml.

 
Role of caveolae- and clathrin-mediated endocytosis in albumin uptake in RLE-6TN cells. We then studied the characteristics of endocytic uptake of albumin by RLE-6TN cells. Endocytosis can be divided into two groups; clathrin-independent and clathrin-mediated endocytosis. The role of caveolae-mediated endocytosis, a clathrin-independent process, was examined. For this purpose, nystatin, indomethacin, and methyl-beta-cyclodextrin, inhibitors of caveolae-mediated endocytosis, were employed. Nystatin at various concentrations did not affect FITC-albumin uptake by RLE-6TN cells (Fig. 8A). No inhibition was observed for indomethacin and methyl-beta-cyclodextrin (Fig. 8, B and C), although the uptake was stimulated at some concentrations of these compounds. Therefore, caveolae-mediated endocytosis is not involved in albumin uptake by RLE-6TN cells.


Figure 8
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  		Fig. 8. Effect of inhibitors of caveolae-mediated endocytosis on FITC-albumin uptake by RLE-6TN cells. A: effect of various concentrations of nystatin on FITC-albumin (20 µg/ml) uptake for 60 min at 37°C. B: effect of various concentrations of indomethacin on FITC-albumin (20 µg/ml) uptake for 60 min at 37°C. C: effect of various concentrations of methyl-beta-cyclodextrin on FITC-albumin (20 µg/ml) uptake for 60 min at 37°C. Each point represents mean ± SE of 3 monolayers. *P < 0.05, significantly different from each control.

 
Next, we examined the role of clathrin-mediated endocytosis by using its inhibitors, phenylarsine oxide and chlorpromazine. The uptake of FITC-albumin was inhibited by the pretreatment of the cells with phenylarsine oxide in a concentration-dependent fashion (Fig. 9A). Similarly, FITC-albumin uptake was inhibited by chlorpromazine (Fig. 9B). In contrast to albumin, FITC-inulin uptake was not affected by phenylarsine oxide at a concentration of 10 µM (data not shown).


Figure 9
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  		Fig. 9. Effect of inhibitors of clathrin-mediated endocytosis on FITC-albumin uptake by RLE-6TN cells. A: effect of various concentrations of phenylarsine oxide on FITC-albumin (20 µg/ml) uptake for 60 min at 37°C. B: effect of various concentrations of chlorpromazine on FITC-albumin (20 µg/ml) uptake for 60 min at 37°C. Each point represents the mean ± SE of 3 monolayers. *P < 0.05, significantly different from each control ({circ}).

 
Furthermore, the effects of potassium depletion and hypertonicity on FITC-albumin uptake, the conditions known to inhibit clathrin-mediated endocytosis (16, 18), were examined. As shown in Fig. 10A, potassium depletion and hypertonicity inhibited FITC-albumin uptake by RLE-6TN cells. On the other hand, these treatments had little or no effect on FITC-dextran uptake (Fig. 10B). Together, these results strongly indicate that FITC-albumin uptake by RLE-6TN cells is mediated by clathrin-mediated but not by caveolae-mediated endocytosis.


Figure 10
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  		Fig. 10. Effect of potassium depletion and hypertonicity on FITC-albumin (A) and FITC-dextran (B) uptake by RLE-6TN cells. Experimental conditions to induce potassium depletion and hypertonicity are described in MATERIALS AND METHODS. A: uptake of FITC-albumin (20 µg/ml) for 60 min at 37°C was measured under the condition of control, potassium depletion, or hypertonicity. B: uptake of FITC-dextran (500 µg/ml) for 60 min at 37°C was measured under the condition of control, potassium depletion, or hypertonicity. Each column represents mean ± SE of 3 monolayers. *P < 0.05, significantly different from each control.

 
Expression of megalin and cubilin and their roles in albumin endocytosis in RLE-6TN cells. The expression of mRNAs of two endocytic receptors, megalin and cubilin, in RLE-6TN cells was examined by RT-PCR. As shown in Fig. 11A, the expression of megalin mRNA was detected in RLE-6TN cells and in the renal cortex. The expression of cubilin mRNA was also detected in RLE-6TN cells at passages 43 and 61 and in the lung and renal cortex (Fig. 11B). Megalin/cubilin-mediated endocytosis is reported to be a Ca2+-dependent process (7). Therefore, the effect of the Ca2+ chelator, EGTA, on FITC-albumin uptake by RLE-6TN cells was examined. The uptake of FITC-albumin was significantly inhibited by the presence of EGTA (Fig. 11C).


Figure 11
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  		Fig. 11. A: expression of megalin mRNA in RLE-6TN cells and in rat renal cortex was analyzed by RT-PCR. The PCR products with [RT(+)] or without [RT(–)] reverse transcription were separated by electrophoresis through 2.0% agarose gels and visualized under ultraviolet light with ethidium bromide. B: expressions of cubilin mRNA in RLE-6TN cells (passages 43 and 61) and in rat lung and renal cortex were analyzed by RT-PCR as described above. C: effect of EGTA on FITC-albumin uptake by RLE-6TN cells. The uptake of FITC-albumin (20 µg/ml) for 60 min at 37°C was measured in the absence (control) or presence of 1 mM EGTA. Each column represents the mean ± SE of 3 monolayers. *P < 0.05, significantly different from control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we examined the uptake mechanisms of FITC-albumin in the cultured alveolar type II epithelial cell line RLE-6TN. When RLE-6TN cells were plated at a density of 10 x 104 cells per 35-mm culture dish, the cells grew with a doubling time of ~28 h (data not shown) and showed epithelial-like morphology (Fig. 6A). The RLE-6TN cells possessed alkaline phosphatase activity (Fig. 1A), a functional marker of differentiated type II cells, although the activity was much lower than that in the lung or isolated alveolar type II cells (12, 33). This finding is comparable with that reported by Driscoll et al. (11), who showed that only an occasional cell was detected expressing alkaline phosphatase activity when a histochemical method was used. The expression of cytokeratin 19 mRNA was detected by RT-PCR analysis in RLE-6TN cells at an early and a late passage used for experiments (Fig. 1B), which is also a marker for differentiated alveolar type II cells (32). These results indicate that the RLE-6TN cells that we employed retain some characteristics of alveolar type II cells.

The uptake of FITC-albumin was almost linear up to 60 min, and showed a marked temperature dependence (Fig. 2). The uptake at 4°C was dramatically lower than at 37°C. The temperature-dependent uptake of FITC-albumin was also confirmed by confocal laser scanning microscopy (Fig. 6, B and C). In addition, the uptake was inhibited by metabolic inhibitors (2,4-dinitrophenol and sodium azide plus 2-deoxy-D-glucose) and by bafilomycin A1 (Fig. 5). Bafilomycin A1 is a specific inhibitor of vacuolar H+-ATPase. Vacuolar H+-ATPase localized in the endosomal membrane is responsible for lowering pH inside the endosome, which is an essential process for the dissociation of ligands and receptors after receptor-mediated endocytosis (30). Inhibition of vacuolar H+-ATPase results in a decreased activity of the receptor-mediated endocytosis (34, 38). Thus these results suggest that FITC-albumin is taken up by RLE-6TN cells by receptor-mediated endocytosis.

The uptake of FITC-albumin by RLE-6TN cells was saturable and was mediated by high- and low-affinity transport systems (Fig. 3). Native (unlabeled) albumin inhibited FITC-albumin (20 µg/ml) uptake by RLE-6TN cells in a concentration-dependent manner (Fig. 4A). Assuming that FITC-albumin at a concentration of 20 µg/ml is taken up by a high-affinity transport system and that the inhibition by native albumin is competitive, the Km (Ki) value of native albumin on FITC-albumin uptake was calculated to be 10.6 mg/ml. This value was much higher than the Km value of the high-affinity FITC-albumin transport system (0.13 mg/ml). Thus the observed uptake system for FITC-albumin may preferentially recognize modified or denatured albumin rather than native albumin, like gp18- or gp30-mediated albumin endocytosis (35). To test this possibility, we synthesized chemically modified albumins (formylated- and maleylated-albumin) and examined their effects on FITC-albumin uptake by RLE-6TN cells. However, the inhibitory potencies of these chemically modified albumins were similar to those of native albumin (Fig. 4B). Therefore, the uptake system is not specific for modified or denatured albumin.

On the other hand, the uptake of FITC-inulin (0.5 mg/ml), a marker of fluid-phase endocytosis, by RLE-6TN cells was not affected by the presence of unlabeled inulin at concentrations of up to 40 mg/ml (data not shown), indicating that the uptake of FITC-inulin is linearly increased with its concentration. The uptake clearance of FITC-albumin (20 µg/ml) was 56.0 ± 1.0 µl·mg protein–1·60 min–1 (mean ± SE of 3 determinations) and was more than 100-fold higher than that of inulin (0.49 ± 0.02 µl·mg protein–1·60 min–1, mean ± SE estimated from 3 inulin concentrations of 0.5, 20, and 40 mg/ml). These results further suggest that albumin uptake by RLE-6TN cells is mediated by receptor-mediated endocytosis.

The intracellular localization of FITC-albumin taken up by RLE-6TN cells was evaluated by confocal laser scanning microscopy. Using LysoTracker red as a lysosomal marker, it was observed that the part of FITC-albumin was localized in lysosomes (Fig. 6, D–F). In other fields of the micrograph, however, FITC-albumin was also observed in compartment(s) other than lysosomes even after 60 min of incubation (Fig. 6, G–I), which may be endosomes containing FITC-albumin.

A fluoroimage analysis after SDS-PAGE showed that FITC-albumin in the cells was gradually degraded over time (~16% at 60 min), although the rest remained intact (Fig. 7). The presence of intact FITC-albumin could be due to continuous endocytosis of intact FITC-albumin into the cells. As the localization of FITC-albumin in lysosomes was observed by confocal laser scanning microscopy, it is likely that FITC-albumin was degraded in the lysosomes.

We next examined the role of caveolae-mediated endocytosis in albumin uptake in RLE-6TN cells. The involvement of caveolae-mediated endocytosis has been suggested in albumin uptake in microvessel endothelial cells and in alveolar type I and type II epithelial cells (20, 21, 23). John et al. (20) examined albumin transport in cultured alveolar type II cells and in isolated rat lung. They showed the involvement of gp60, an albumin-binding glycoprotein, in albumin transport and showed that filipin, a caveolae disrupting agent, blocked the transport. The important roles of gp60 and caveolae in albumin endocytosis were also reported and are well known in microvessel endothelial cells (21, 42). Caveolae, which are flask-shaped invaginations of the plasma membrane, are cholesterol- and sphingolipid-rich microdomains. The shape and structure of caveolae are conferred by caveolin, a dimeric protein that binds cholesterol (9, 31). In this study, we employed nystatin, indomethacin, and methyl-beta-cyclodextrin as inhibitors of caveolae-mediated endocytosis. Nystatin and methyl-beta-cyclodextrin inhibit caveolae-mediated endocytosis by interacting with cholesterol in the plasma membrane (1, 36), and indomethacin inhibits the process by inhibiting the internalization of caveolae and the return of plasmalemmal vesicles to the cell surface (37, 41). However, albumin uptake by RLE-6TN cells was not inhibited by these inhibitors (Fig. 8). The presence of caveolae or the expression of caveolin in alveolar type II cells is controversial (6, 20). Campbell et al. (6) showed that caveolin-1 was not expressed in freshly isolated rat type II cells but was expressed when type II cells were transdifferentiated to a type I-like phenotype. On the other hand, John et al. (20) showed the presence of caveolin-1 in cultured alveolar type II cells obtained from rat lung, using anti-caveolin-1 monoclonal antibody. Therefore, we examined caveolin-1{alpha} mRNA expression in RLE-6TN cells by RT-PCR, and its expression was observed (data not shown). Thus RLE-6TN cells may have a caveolae-mediated endocytic pathway, but that pathway is not involved in FITC-albumin uptake.

Clathrin-mediated endocytosis is a better-understood endocytosis (9). Clathrin-mediated endocytosis occurs constitutively in all mammalian cells and carries out continuous uptake of nutrients, such as cholesterol-laden low-density lipoproteins and iron-laden transferrins that bind to their own receptors. Clathrin-mediated endocytosis involves the concentration of transmembrane receptors and their bound ligands into coated pits on the plasma membrane (9). The coated pits are formed by the assembly of cytosolic coat proteins, the main assembly unit being clathrin. Here, we examined the effect of clathrin inhibitors, phenylarsine oxide and chlorpromazine, on albumin uptake in RLE-6TN cells. Phenylarsine oxide inhibits clathrin-mediated endocytosis by reacting with vicinal sulfhydryls to form stable ring structures (38, 41). Chlorpromazine inhibits the process by inducing the loss of coated pits from the cell surface, probably by interacting with AP-2 binding to membranes (43). Both compounds inhibited FITC-albumin uptake by RLE-6TN cells in a concentration-dependent manner (Fig. 9), indicating the involvement of clathrin-mediated endocytosis. In contrast, these compounds did not affect FITC-inulin uptake in RLE-6TN cells (data not shown). Furthermore, the effects of potassium depletion and hypertonicity on FITC-albumin uptake by RLE-6TN cells were examined. These treatments are known to inhibit clathrin-mediated endocytosis by inducing the disappearance of clathrin-coated pits from the plasma membrane (16, 18). Both treatments inhibited FITC-albumin uptake by RLE-6TN cells (Fig. 10A), whereas they hardly affected FITC-dextran uptake (Fig. 10B), a process mediated by fluid-phase endocytosis. Together, these results strongly suggest that FITC-albumin uptake by RLE-6TN cells is mediated by clathrin-mediated but not by caveolae-mediated endocytosis.

At this stage, information concerning the mechanisms of albumin uptake in alveolar type II cells is quite limited. As far as we know, there is no report showing the involvement of clathrin-mediated endocytosis in albumin uptake in type II cells. On the other hand, in the kidney, megalin and cubilin are known to play an important role in the clathrin-mediated endocytosis of filtered proteins, including albumin, from the tubular lumen (2, 7, 10, 26). Megalin, a member of the low-density lipoprotein receptor gene family, is an endocytic receptor that recognizes a large number of protein ligands as well as aminoglycoside antibiotics and is abundantly expressed and located in clathrin-coated pits in the apical membrane of renal proximal tubular epithelial cells (7, 14, 28, 29). Cubilin is also a multiligand endocytic receptor coexpressed with megalin in renal proximal tubules, although its structure is quite different from megalin and has no transmembrane domain (25). Therefore, megalin is assumed to mediate internalization of cubilin and its ligands bind to cubilin (7, 15). Recently, megalin and cubilin were shown to be expressed in alveolar type II cells (24). Therefore, we examined the expression of megalin and cubilin mRNAs in RLE-6TN cells by RT-PCR and found that these two receptors are expressed also in RLE-6TN cells (Fig. 11, A and B).

In most cases, ligand binding to megalin is Ca2+ dependent. In addition, the binding between megalin and cubilin is also reported to be Ca2+ dependent (7, 8, 27). Therefore, Ca2+ dependence of albumin uptake was examined in RLE-6TN cells. The uptake of albumin was significantly suppressed in the presence of EGTA (Fig. 11C), suggesting that albumin uptake is Ca2+ dependent, similar to the case in megalin-mediated ligand uptake. These results, together with information in the literature, may indicate the involvement of megalin/cubilin, at least partially, as receptors for albumin endocytosis in RLE-6TN cells.

On the other hand, as described above, the apparent affinity of native albumin for the endocytic system in RLE-6TN cells seemed to be fairly low (Km value estimated to be 10.6 mg/ml or 158 µM). Although the affinity of albumin to megalin has not been reported yet as far as we know, the Kd value of albumin to purified cubilin was reportedly 0.63 µM (2). Thus there is a great difference between the apparent affinities of albumin to the endocytic system observed in the present study and to cubilin, suggesting little or no contribution by cubilin. In any case, the contribution of megalin/cubilin for albumin endocytosis in RLE-6TN cells needs to be studied further with, for example, short interfering RNAs that knock-down these receptors.

The involvement of a clearance receptor(s) specific for modified or denatured albumin like gp18 or gp30 is also unlikely because the inhibitory potencies of chemically modified albumins were similar to those of native albumin. In addition, gp18- and gp30-mediated endocytosis of albumin is reportedly a caveolae-mediated pathway (3). Another possibility is the involvement of gp60, an endocytic receptor for albumin well studied in microvessel endothelial cells. John et al. (21) reported that 125I-labeled albumin is taken up by microvessel endothelial cells with high-affinity (Kd1 = 0.87 µM) and low-affinity (Kd2 = 93.3 µM) systems. These Kd values were fairly compatible with observed Km values for FITC-albumin in the present study. However, they also showed that 125I-labeled albumin uptake, probably mediated by gp60, was inhibited by the disruption of caveolae with the use of methyl-beta-cyclodextrin, in contrast to the present study. Thus certain characteristics of the FITC-albumin uptake in RLE-6TN cells observed in the present study cannot be well explained by known receptors.

The apparent affinity of native albumin for the endocytic system in RLE-6TN cells was fairly low (Km value of 10.6 mg/ml, as described above). Such a low affinity may be suitable for the clearance of albumin from the alveolar surfaces not only under normal conditions but also in such diseased states as pulmonary edema, because the concentration of albumin in alveolar fluid is low under normal conditions but increases to 40–65% of plasma level (~40 mg/ml) in hydrostatic pulmonary edema and to 75–95% in lung injury pulmonary edema (17). Thus one of the roles of the endocytic system observed in the present study may be the clearance of albumin from alveolar fluid under physiological and pathophysiological conditions.

In conclusion, we examined uptake mechanisms of FITC-albumin using the cultured alveolar type II epithelial cell line RLE-6TN. The uptake of FITC-albumin in RLE-6TN cells was found to be mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and FITC-albumin taken up by the cells was gradually degraded in lysosomes. Further studies will be needed to identify the main receptor(s) involved in the endocytosis, and the physiological/pathophysiological roles of this endocytic system.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Takano, Dept. of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima Univ., 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan (e-mail address: takanom{at}hiroshima-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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