Apical location of ferroportin 1 in airway epithelia and its role in iron detoxification in the lung
Funmei Yang,1
David J. Haile,2
Xinchao Wang,3
Lisa A. Dailey,3
Jacqueline G. Stonehuerner,3 and
Andrew J. Ghio3
Departments of 1Cellular & Structural Biology and 2Medicine, The University of Texas Health Science Center, San Antonio, Texas; and 3National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, North Carolina
Submitted 8 December 2004
; accepted in final form 27 February 2005
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ABSTRACT
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Ferroportin 1 (FPN1; aka MTP1, IREG1, and SLC40A1), which was originally identified as a basolateral iron transporter crucial for nutritional iron absorption in the intestine, is expressed in airway epithelia and upregulated when these cells are exposed to iron. Using immunofluorescence labeling and confocal microscopic imaging techniques, we demonstrate that in human and rodent lungs, FPN1 localizes subcellularly to the apical but not basolateral membrane of the airway epithelial cells. The role of airway epithelial cells in iron mobilization in the lung was studied in an in vitro model of the polarized airway epithelium. Normal human bronchial epithelial cells, grown on membrane supports until differentiated, were exposed to iron, and the efficiency and direction of iron transportation were studied. We found that these cells can efficiently take up iron across the apical but not basolateral surface in a concentration-dependent manner. Most of the iron taken up by the cells is then released into the medium within 8 h in the form of less reactive protein-bound complexes including ferritin and transferrin. Interestingly, iron release also occurred across the apical but not basolateral membrane. Our findings indicate that FPN1, depending on its subcellular location, could have distinct functions in iron homeostasis in different cells and tissues. Although it is responsible for exporting nutrient iron from enterocytes to the circulation in the intestine, it could play a role in iron detoxification in airway epithelial cells in the lung.
iron metabolism; divalent metal transporter-1; ferritin; transferrin
RECENT DISCOVERY OF KEY PROTEINS involved in iron transport across the cell membrane has opened up a new era of research on the roles of these proteins in iron absorption in intestinal cells and iron metabolism in other cells. Among these proteins are two transmembrane iron transporters, divalent metal transporter (DMT1; aka DCT1, Narmp2, and SLC11A2) and ferroportin 1 (FPN1; aka MTP1, IREG1, and SLC40A1). In most proliferating cells, iron is taken up by the transferrin-transferrin receptor pathway. In the duodenal epithelial cells, in which transferrin receptor is absent, DMT1 is responsible for nonheme iron uptake from the intestinal lumen (12, 15, 27) and FPN1 is responsible for iron's exit to the circulation (1, 9, 20). In accord with the proposed functions in these cells, DMT1 is located mainly on the apical and FPN1 on the basolateral membrane of the enterocytes (1, 5, 9, 20). In addition to their roles in iron absorption, DMT1 is also responsible for transferrin-associated endosomal iron transport in erythroid precursors, and FPN1 is required for iron recycling in reticuloendothelial system (RES). The Belgrade (b) rats, which have a mutation at exactly the same amino acid residue on DMT1 gene as the microcytic anemic (mk) mice, have defects in both erythroid iron utilization and intestinal iron uptake (11). Mutations in the FPN1 gene lead to iron overload (8, 21, 22) presumably due to reduced iron export from cells, particularly RES cells. Because iron homeostasis is crucially important in all living organisms, iron metabolism is tightly controlled to ensure an adequate supply without excessive accumulation of this essential but potentially toxic metal. This can be demonstrated at the molecular level in the regulation of DMT1 and FPN1. Expression of these two key transporters is regulated by body iron store and dietary iron (5, 20, 37, 38), erythroipoietic activity (20), and mediators for inflammation (18, 31, 35). In addition to enterocytes and RES cells, DMT1 and FPN1 have been detected in many other cells and tissues, including those in the kidney, brain, and lung. The physiological functions of DMT1 and FPN1 in these cells remain largely unknown.
We have recently found that DMT1 and FPN1 are produced at high levels in lung airway epithelial cells and alveolar macrophages (14, 30, 36). With exposure to iron, synthesis of DMT1 and FPN1 increased in these cells in both humans and rodents. Alveolar macrophages have long been known to be important in iron scavenging in the lower respiratory tract, and it is not surprising that DMT1 and FPN1 are expressed in these cells. It is intriguing that both of these iron transporters are produced in airway epithelial cells in a manner similar to what was observed in duodenal epithelial cells. If the airway epithelium is to participate in host defense against environmental invaders, then the presence of key iron transporters in these cells may not be for nutrient iron absorption. In fact, iron toxicity has been implicated in the pathogenesis of several lung diseases, including adult respiratory distress syndrome (4, 6), bronchopulmonary dysplasia (10, 23), and lung inflammation induced by toxic mineral dusts (17, 25). Catalytically active iron released from inhaled pollutants or injured tissues can cause oxidative damage and contribute to subsequent clinical complications in these disorders. As part of lung defense, alveolar macrophages could participate in iron scavenging, and airway epithelial cells could also be important in iron detoxification.
In our previous studies, we have found that human bronchial epithelial cells are capable of taking up a large amount of iron (30). We demonstrated that nontransferrin-bound iron (NTBI) uptake by these cells was associated with DMT1. With exposure to iron, both DMT1 expression and transport of this metal into the cells increased (30). In the lung of Belgrade rats, the efficiency of iron uptake by lung cells was significantly reduced (Ghio AJ, unpublished results). These animals also showed increased lung injury when exposed to iron-containing particles, which suggested an association of DMT1 with iron detoxification in the lung. In the current paper, we report our novel findings of the unique localization of FPN1 on the apical, but not the basolateral, membrane of airway epithelial cells in human and rodent lungs. We demonstrate that differentiated, polarized bronchial epithelial cells have a high capacity to take up iron and then release most of the newly imported iron in stable protein-bound complexes. Both iron uptake and release in these cells are polarized and occur only on the apical and not basolateral membrane. Our results indicate that FPN1, depending on its subcellular locations, could have distinct functions in iron metabolism in different tissues. Although FPN1 is necessary for exporting nutrient iron into the circulation in the intestinal epithelium, it likely plays an important role in iron detoxification in the airway epithelium. Together with DMT1, it participates in a conversion of catalytically active iron into less-toxic protein-bound iron complexes in airway epithelial cells.
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MATERIALS AND METHODS
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Culture of human bronchial epithelial on membrane supports.
Primary normal human bronchial epithelial (NHBE) cells were obtained from healthy, nonsmoking adult volunteers after consent was obtained. The protocol and consent form were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. Cells were obtained by cytological brushing at bronchoscopy and expanded to passage 3 in bronchial epithelial growth medium (BEGM). They were plated on collagen-coated filter supports inserted into 12-well culture plates and maintained as described previously (28). Retinoic acid was added on day 2 (once cells reached 100% confluence) to promote differentiation. Air-liquid interface was created on day 6 by removing the apical medium. Thereafter, basal medium was replaced every 48 h. The cells were maintained until they had uniformly differentiated into ciliated, mucus producing cells
10 days later.
Culture of BEAS-2B cells.
BEAS-2B cell line is an immortalized line of NHBE cells derived by transfection of primary cells with SV40 early-region genes. This particular subclone undergoes squamous differentiation in response to serum (16). Cells were grown to 90100% confluence on uncoated plastic 12-well plates in Clonetics keratinocyte growth medium (Cambrex Bio Science, Walkersville, MD), which is Clonetics keratinocyte basal medium supplemented with SingleQuotes containing human epidermal growth factor, bovine insulin, hydrocortisone, bovine pituitary extract, and Gentamicin sulfate amphotericin. Previous investigation has demonstrated no differences in the response of BEAS-2B cells to iron relative to NHBE cells (13).
Immunofluorescence studies.
Surgical specimens of human lung and normal mouse lung tissues were quick-frozen in Tissue-Tek optimum cutting temperature compound (Miles, Elkhart, IN) and stored at 80°C until used for the preparation of frozen sections. Alveolar macrophages isolated from lung lavage were allowed to adhere to Lab-Tek chamber slide (Miles) during overnight incubation at 37°C in DMEM plus 10% fetal calf serum in a CO2 incubator. Lung sections (5 µm) and cells on slides were fixed with 4% paraformaldehyde (PFA) in 1x PBS, pH 7.4, at 4°C for 20 min. After being washed with PBS several times, the specimens were treated with 0.5% Triton in 1x PBS at room temperature for 610 min, washed in PBS, and incubated at room temperature in 1.5% normal goat serum in PBS for 30 min. Samples were then incubated with primary antibodies including an anti-mouse FPN1 antibody (1) and in some experiments antitransferrin receptor (Pharmingen International, San Diego, CA) in PBS containing 1% fat-free BSA. After a 30-min incubation at room temperature, the slides were washed with PBS containing 1% BSA and incubated for 30 min with anti-rabbit IgG conjugated with FITC-Alex488 (Molecular Probes, Eugene, OR) and/or anti-mouse IgG conjugated with Tritc (Zymed Laboratories, San Francisco, CA) in PBS plus 1% BSA. The slides were washed with PBS, and in some cases nuclei were stained with propidium iodine. Slides were washed again in PBS, covered with Vectashield (Vector Laboratories, Burlingame, CA) cover slides, and stored in the dark. Images were captured on an Olympus FV-500 confocal microscope.
Measurement of iron concentrations.
Iron concentrations were quantified using inductively coupled plasma atomic emission spectroscopy (ICPAES) at a wavelength of 238.204 (model P30; Perkin Elmer, Norwalk, CT). Single element standards were used to calibrate the instrument (Fisher, Pittsburgh, PA). The limit of detection approximated 10 ppb. Samples were analyzed for 57Fe on a Perkin Elmer Elan 6000 inductively coupled plasma mass spectrometer (ICPMS) with a Scott cross-flow nebulizer under cool plasma conditions to minimize the formation of oxides. The settings used were: power = 700 W, nebulizer flow = 1.3 l/min, lens = 3 V (static). Co was added at a level of 50 ppb as an internal standard using an online internal standard addition kit (Perkin Elmer part no. N0690673).
Measurement of total cellular nonheme iron concentrations.
NHBE cells were grown in BEGM, which contains a physiological concentration of iron
10 µM, on Transwells until differentiated. The medium was removed, both the apical and basolateral chambers were washed with 1.0 ml of Hanks' buffered salt solution (HBSS), and 1.0 ml of HBSS was placed in the basolateral chamber. Twenty-five microliters of HBSS varying in concentration from 0 to 2,000 µM ferric ammonium citrate (FAC) or 57FeCl3 was then added to the apical chamber. Alternatively, various concentrations of FAC were included in the basolateral chamber. To determine iron uptake efficiency, the membrane with the cells was removed from its support after 4 h, washed three times in HBSS, placed in 1.0 ml of 3 N HCl containing 10% trichloroacetic acid, and hydrolyzed at 70°C for 18 h. After centrifugation at 600 g for 10 min, the concentration of iron in the supernatant was determined by ICPAES, and the concentration of 57Fe was determined with the ICPMS. For iron release experiments, cells were exposed to iron for 4 h, the apical chamber was washed three times in HBSS, and 1.0 ml of HBSS in the basolateral chamber was replaced. After 8 h, the membrane with the cells was removed from its support, washed, and hydrolyzed, and nonheme iron that remained in the cells was measured as described above.
Measurements of iron released into apical or basolateral chambers.
NHBE cells were grown in BEGM on Transwells until differentiated. Cells were exposed to FAC or 57Fe for 4 h and allowed to release iron as described above. After 112 h of release, 1.0 ml of HBSS was added to the apical chamber and immediately removed. The basolateral fluid was also taken. After centrifugation at 600 g for 10 min, the concentrations of iron or 57Fe in these supernatants were determined by ICPAES or ICPMS.
Measurement of membrane-bound iron concentrations.
The membrane with the cells was removed from its support and washed with HBSS twice. Surface-bound iron was then removed by incubating the cells in 1.0 ml of 140 mmol/l NaCl, 10 mmol/l PIPES, 5.0 mmol/l sodium dithionite, and 5.0 mmol/l bathophenanthroline disulfonic acid. The supernatant was removed and centrifuged at 600 g for 10 min. Iron in the supernatants was measured using ICPAES.
Immunoprecipitation.
For immunoprecipitation, NHBE or BEAS cells were exposed to 500 µM FAC for 4 h, washed, and allowed to release iron during the subsequent 8 h of incubation as described above. The medium was collected and precipitated with antiferritin, antitransferrin, or control IgG in the presence of protein A/G agarose. After centrifugation at 600 g for 10 min, the amounts of iron that remained in the clear supernatants were measured by ICPAES as described in Measurement of iron concentrations.
Statistics.
Data is expressed as mean values ± SE. Differences between multiple groups were compared using one-way analysis of variance (7) with Scheffé's post hoc test. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.
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RESULTS
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Subcellular localization of FPN1 in human lung cells.
Cellular distribution and intracellular location often provide evidence to support the functions of the protein. Using immunohistochemical and in situ hybridization techniques, we have previously identified airway epithelial cells and alveolar macrophages as two major cell types that express FPN1 gene (36). To define the subcellular locations of FPN1, immunofluorescent labeling with an anti-FPN1 antibody and a secondary antibody conjugated with FITC (green fluorescence) was conducted in human frozen lung sections and visualized by confocal microscopy. As shown in low magnification (Fig. 1), fluorescent labeling for FPN1 localizes mainly to the airway epithelium. No significant specific labeling was seen in other lung tissues including the alveolar epithelium, smooth muscle, blood vessels, and connective tissues. The distribution of the labeling in airway epithelia is restricted to the apical region facing the lumen (Figs. 1 and 2B). At higher magnification (Fig. 2, CE), it was clear that FPN1 localizes to the apical membrane but not to either the basolateral membrane or cytoplasm of these cells. A low level of fluorescent labeling can be seen in the nucleoli of the airway epithelial cells. Because FPN1 contains 12 transmembrane regions and is membrane bound, it cannot be localized to the nucleolus. In addition, we have not observed any nucleolar localization of FPN1 with overexpression of FPN1 in various tissue culture cells (Liu XB, Yang F, and Haile DJ, unpublished results). The nucleolar staining is likely an artifact due to another antigen that cross-reacts with the antibody. For alveolar macrophages, which could not be clearly identified in human frozen lung sections, an immunofluorescence labeling experiment was conducted on freshly isolated lavage cells adhered onto chamber slides. In contrast to what observed in airway epithelial cells, FPN1 in these macrophages was localized mainly to cytoplasmic organelles and to a much lesser extent on the cell membrane (Fig. 2F). The identities of these subcellular structures are being investigated and will be reported elsewhere.

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Fig. 1. Localization of ferroportin 1 (FPN1) in human lung by immunofluorescent staining. A human lung section was stained with an anti-FPN1 antibody and a secondary antibody conjugated with FITC (green fluorescence). Nuclei were stained with propidium iodide (red fluorescence), and the image was captured by a confocal microscope. FPN1 is localized mainly to the airway epithelium (arrows). No significant staining was detected in other regions in the lung including the alveolar region (av). *Airway lumen. Original magnification: x200.
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Fig. 2. Subcellular localization of FPN1 in human airway epithelial cells and alveolar macrophages. Human lung sections (BE) and alveolar macrophages (F) were stained with an anti-FPN1 antibody and a secondary antibody conjugated with FITC (green fluorescence). The control lung section (A) was reacted with IgG. Nuclei (in AE) were stained with propidium iodide (red fluorescence). Arrows point to airway epithelial cells; the lumen is marked by an asterisk. In airway epithelia, FPN1 is localized mainly to the apical membrane that can be clearly identified in higher magnifications (CE), although some cross-reactive fluorescence can be seen in the nucleoli. In alveolar macrophages (F), FPN1 is present in cytoplasm membrane and intracellular vesicles, but not in nuclei (n). Original magnifications: A and B, x200; C, x400; D, x1,200; E, x2,400; F, x2,000.
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Subcellular localization of FPN1 in mouse lung cells.
Localization of FPN1 in mouse lung cells was studied using frozen lung sections and freshly isolated alveolar macrophages. In Fig. 3, the fluorescent signal for anti-FPN1 is localized mainly to the apical membrane of the airway epithelial cells. Consistent results were obtained in experiments in which transferrin receptor was used as a marker (Fig. 3B) or the nuclei were stained with propidium iodine (Fig. 3, C and D). In both experiments, the fluorescent signal for FPN1 can be seen as short, discontinuous, sharp lines on the apical membrane of airway epithelial cells. In freshly isolated alveolar macrophages (Fig. 3, E and F), FPN1 was detected mainly in a cytoplasmic organelles similar to what was seen in human alveolar macrophages. Our data from both human and rodent lungs suggest that FPN1 may mediate iron export from airway epithelial cells across the apical membrane to the airway lumen.

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Fig. 3. Subcellular localization of FPN1 in mouse airway epithelial cells and alveolar macrophages. Mouse lung sections (BD) and alveolar macrophages (E and F) were stained with an anti-FPN1 antibody and a secondary antibody conjugated with FITC (green fluorescence). B was stained with anti-transferrin receptor (CD71) in addition to anti-FPN1, whereas the control section (A) was stained with antitransferrin receptor and normal rabbit IgG. A second antibody conjugated with Tritc (red fluorescence) was used to react with antitransferrin receptor. The nuclei in CE were stained with propidium iodide (red fluorescence). FPN1 is localized to the apical membrane of airway epithelial cells (arrows) that can be visualized as sharp lines in BD and is present in plasma membrane as well as intracellular vesicles (E and F) in alveolar macrophages. The airway lumen is marked by an asterisk, and a nucleus (n) is marked in F. Original magnifications: AD, x1,200; C, x1,500; D, x2,000.
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Bronchial epithelial cells take up and release iron in a concentration-dependent manner.
In our previous investigations, we have shown that the expression of both DMT1 and FPN1 in human bronchial epithelial cells, which were derived from the upper airway epithelium, increased when exposed to iron in a dosage-dependent manner (30, 36). In this study, we used an in vitro model of the bronchial epithelial cells to determine whether the efficiency of these cells to take up and/or release iron is also increased when exposed to the metal. NHBE cells were grown on membrane supports in a liquid-air interface until differentiated as described previously (28). A small volume of iron solution, which contained either FAC or 57Fe-labeled ferric chloride, was then applied to the apical surface of the cells as described in MATERIALS AND METHODS. The efficiency of iron uptake and its subsequent release were measured. The use of labeled and unlabeled iron allowed us to monitor the movement of newly acquired iron and total iron including endogenous cellular iron, respectively, during the experiments. Exposure of cells to iron for 4 h resulted in an increase in total cellular iron (Fig. 4, A and B) in a manner that was dependent on the initial concentrations of iron added to the apical chamber. A linear increase in total cell iron was observed when the cells were exposed to 02,000 µM iron. To study the ability of these cells to release iron after an uptake, we washed and incubated cells on membrane support with fresh iron-free buffer in the basolateral chamber for an additional 8 h. Total cell iron after release was measured and shown (Fig. 4, A and B) as a function of initial iron concentration added to the cells. The amount of iron released, which is the net difference between total cell iron after uptake and after release, is shown in Fig. 4, C and D. This experiment revealed that bronchial epithelial cells are very efficient in releasing iron. This was particularly clear when 57Fe-labeled iron was used in the assay (Fig. 4, B and D), which demonstrated that very little of the 57Fe that was transported into the cells remained in the cells after release when the initial added iron was not higher than 1,000 µM (Fig. 4B). Even in cells exposed to a concentration of iron as high as 2,000 µM, >65% of the iron taken up by the cells was released within 8 h during the subsequent incubation. Because the amounts of iron released from cells exposed to FAC and 57Fe are very much the same (Fig. 4, C and D), it was also evident that there was no significant mobilization of the iron pool originally present in the cells. In addition, no significant change in cell morphology was observed during the course of the experiments.

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Fig. 4. Concentration-dependent iron uptake and release in differentiated human bronchial epithelial cells. Normal human bronchial epithelial (NHBE) cells were grown on Transwells until differentiated and exposed to 02,000 µM ferric ammonium citrate (FAC; A, C, E, and F) or 57Fe (B and D) for 4 h, and total nonheme cell iron was measured to determine iron uptake ( in A and B). For iron release experiments, cells were exposed to iron for 4 h, washed, and incubated for an additional 8 h in fresh iron-free buffer before the remaining cell iron was measured ( in A and B). The amount of iron released ( in C and D) was computed by subtracting cell iron after release from that after uptake. The membrane-bound iron (E), which was measured after uptake, did not demonstrate a concentration dependent increase. Iron was added to the apical chamber in AE, whereas it was added to either the apical or basolateral chamber in F for the study of polarity-dependent iron uptake. *P < 0.05 compared with cells exposed to buffer alone.
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In our previous study using submerged cultures, we have shown that iron was truly transported into bronchial epithelial cells. The accuracy of the methodology for measuring cellular iron uptake and release has been established by measuring the quenching of internalized fluorescent calcein and the iron bound reversibly to surface anionic sites on the cells (30). In this study, the membrane-bound iron was measured after the cells were exposed to 02,000 µM iron for 4 h. Although there were small, measurable concentrations of membrane-associated iron, the amount did not change with exposures to increasing concentrations of iron (Fig. 4E). In contrast, total nonheme iron in the cells did increase with the concentration of iron to which the cells were exposed. Therefore, the iron released from the cells derived mainly from that which was transported inside the cells.
To determine whether the ability of the differentiated airway epithelial cells to mobilize iron is polarity dependent, we compared iron uptake across apical and basolateral surfaces. NHBE cells grown on membrane supports were exposed to 0500 µM of FAC added either in the apical or basolateral chambers. Total cell iron after a 4-h exposure was measured and compared (Fig. 4F). Although there was a dosage-dependent increase of iron uptake in cells in which iron was given in apical chamber, there was no significant iron uptake in cells in which iron was given in the basolateral chamber up to 250 µM. When the cells were exposed to 500 µM FAC, iron uptake across the basolateral surface could be detected. However, the amount of iron taken up from the basolateral surface was only
25% of that from the apical surface under this same condition. It is evident that iron uptake predominantly occurs across the apical membrane in these cells.
Iron is released from the apical but not basolateral surface of bronchial epithelial cells.
To determine whether iron was released across the apical or basolateral membrane, NHBE cells were exposed to 02,000 µM of iron for 4 h, and iron released into the apical and the basolateral chambers during the subsequent incubation was collected and measured as described in MATERIALS AND METHODS. In cells exposed to <1,000 µM of FAC or 57Fe (Fig. 5, A and B), iron was recovered mainly from the apical surface, and no significant amount of iron was detected in the basolateral chamber after release. When the cells were incubated with a concentration of iron >1,000 µM during uptake, the amount of iron released into the basolateral chamber became significantly increased. Nevertheless, <20% of iron released by the cells was detected in the basolateral chamber in cells exposed to a concentration of iron as high as 2,000 µM. The presence of iron in the basolateral chamber may have resulted from iron toxicity-induced cell damage, as will be discussed.
We also studied the kinetics of iron release. Cells were exposed to 1,000 µM of FAC for 4 h, and iron released across apical and basolateral surface at different times during the subsequent incubation was measured (Fig. 5C). About 70% of the total iron released was recovered within 4 h during the subsequent incubation. By 8 h of incubation, most of the iron was released. Again, iron released was recovered from the apical chamber. No significant amount of iron was detected in the basolateral chamber even after a prolonged incubation up to 12 h.
Iron is released by bronchial epithelial cells as protein-bound complexes.
To investigate the possible biological functions of the iron mobilization observed in bronchial epithelial cells, we analyzed the forms of the iron release by these cells after an initial uptake. One of the candidate means of iron release was the iron storage protein ferritin, which has been detected in human sputum (3). In a previous study, Ghio et al. (13) found that bronchial epithelial cells grown in submerged culture can synthesize ferritin. The level of ferritin expressed in these cells was greatly increased when following exposure to iron. Synthesis and release of ferritin were also demonstrated in iron-loaded alveolar macrophages from smokers (32) and in Kupffer cells after the ingestion of red blood cells (26). To determine whether ferritin accounts for the iron released in our experimental model of airway epithelia, NHBE cells were exposed to 500 µM of FAC, as described in the section Bronchial epithelial cells take up and release iron in a concentration-dependent manner, for 4 h, washed, and allowed to release iron during an additional 8 h of incubation. When iron recovered from the apical chamber was immunoprecipitated with antiferritin,
6065% of the iron was retained in the supernatant (Fig. 6), and 3540% of the iron was coprecipitated with ferritin, whereas no significant amount of iron was precipitated when normal rabbit IgG was used for precipitation.

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Fig. 6. Iron was released as ferritin-bound complex by human bronchial epithelial cells grown in membrane supports. NHBE cells grown in membrane supports were exposed to 500 µM FAC for 4 h, washed, and allowed to release iron for 8 h. Iron released was precipitated with either anti-human ferritin (AB to ferritin) or normal IgG. The amount of iron retained in the supernatant was compared with the sample without any treatment. *P < 0.05 compared with sample without treatment.
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Because ferritin-associated iron only accounted for 3540% of all the iron released by NHBE cells, we investigated whether the iron released could be bound to other proteins. In the intestine, iron taken up by duodenal epithelial cells is believed to exit to circulation as transferrin-bound iron. Airway epithelial cells are capable of synthesizing transferrin (33) in addition to ferritin. We therefore conducted a similar experiment to determine whether iron was also released as transferrin bound. In this experiment, we used a human bronchial epithelial cell line, BEAS-2B, grown in submerged culture. This cell line is more readily available and has been shown to behave similarly to the primary culture of NHBE cells in all the many aspects of iron metabolism including iron uptake, iron release, and the secretion of ferritin (13, 30). The use of this submerged culture also facilitated the recovery of the released iron in a single well of medium. Cells were exposed to 500 µM of FAC in serum-free HBSS for 4 h, washed, and allowed to release iron in HBSS during the subsequent incubation. When the medium collected after 8 h of release was precipitated with antiferritin,
40% of the iron was coprecipitated with ferritin, which was similar to what was observed in NHBE cells grown on membrane supports. When the same medium was precipitated with antitransferrin,
3540% of the iron was coprecipitated with transferrin (Fig. 7A). Again, no significant amount of iron was precipitated when the sample was treated with normal IgG (data not shown). These results indicated that ferritin and transferrin are the two major forms of iron released by bronchial epithelial cells. Interestingly, when the media collected at different time during release were compared, it revealed that there was more transferrin than ferritin in the first few hours of release (Fig. 7A). As the amount of transferrin decreased during the subsequent incubation, the amount of ferritin began to increase. Consistent with our findings, total iron released also increased as a function of time in this experiment (Fig. 7B). Nevertheless, transferrin and ferritin consistently accounted for 80% or greater of the iron released at all time points sampled during the 8 h of incubation.

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Fig. 7. Release of iron as ferritin- and transferrin-bound forms by human bronchial epithelial BEAS-2B cells. BEAS-2B cells grown in submerged culture were exposed to 500 µM FAC for 4 h, washed, and allowed to release iron for 8 h. Iron released was precipitated with either anti-human ferritin or anti-human transferrin. The amount of iron coprecipitated with each antibody at each time point is shown in A as a percentage of total iron released, and the total iron released to the medium at each time point is shown in B.
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DISCUSSION
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Since its discovery, FPN1 has been detected in duodenal mucosa, placental syncytiotrophoblast, splenic macrophages, and Kupffer cells in the liver. Although the presence of FPN1 in these cells, which all play important role in iron mobilization, is consistent with its proposed function of exporting iron from the cells, the role of FPN1 in iron homeostasis has only begun to be understood. It is believed that FPN1 is a universal exporter for iron in several different tissues. Based on the searches of protein sequence databases, which identified six different species forms of FPN1 with no other proteins showing significant homology, McKie and Barlow (19) concluded that FPN1 is the sole member of the SLC40 transporter family. In polarized duodenal epithelial cells, FPN is localized mainly to the basolateral membrane. It has been postulated that FPN1, which works together with hephastin (a ferroxidase), translocates iron across the basolateral membrane into the circulation as a transferrin-bound form. In the placenta, FPN1 is present in the basolateral, but not apical, membrane of the syncytiotrophoblast where the iron is transferred from the maternal circulation to the fetus. In reticuloendothelial cells, FPN1 is present in cytoplasm membrane as well as the membrane of intracellular vesicles (1, 35). FPN1-mediated iron export is likely responsible for the recycling of iron from the breakdown of heme from senescent erythrocytes. FPN1 mutations have been reported in many countries regardless of ethnicity and appear to be the most common cause of hereditary iron overload beyond HFE hemocromatosis (reviewed in Ref. 24). Our finding that FPN1 is expressed in airway epithelial cells, whose function is neither nutritional absorption nor iron reutilization, raises intriguing questions on the direction of iron export and the physiological functions of FPN1 in these cells. We demonstrated that FPN1 is located uniquely on the apical but not basolateral membrane of these cells in both human and rodent lungs. This is consistent with our finding that polarized bronchial epithelial cells export iron across the apical but not basolateral membrane in an in vitro model of the airway epithelium. The mechanism involved in the iron transport mediated by FPN1 is not known, nor has FPN1 binding to iron been demonstrated. Nevertheless, our study suggests that depending on its subcellular locations, FPN1 can modulate the direction of iron export from the cells. In the duodenal epithelium, FPN1 transports iron across the basolateral membrane to the adjacent tissues/circulation. Although a study using knockout/knockdown technology is needed for definite proof of the function of FPN1 in airway epithelia, FPN1 is the only known cellular iron exporter that may potentially mediate the release of iron across the apical membrane to the airway lumen. FPN1 may therefore play a key role in iron absorption, iron recycling, or iron detoxification in different tissues.
The present study and results from our previous work provide several lines of evidence for DMT1 and FPN1 being two critical components of the iron transport system in lung airway epithelia. First, expression of these two proteins in the lung is cell-type specific. They are present at a high level in airway epithelial cells but not other lung cells except for alveolar macrophages. Second, expression of these two proteins was upregulated in airway epithelial cells in culture and in experimental animals when exposed to iron in a dosage-responsive manner (30, 36). In addition, we also have unpublished preliminary data showing that the expression of FPN1 was increased in human bronchial epithelial cells within 4 h following exposure to iron. In contrast, transferrin receptor is downregulated when the cells are exposed to iron. Third, the efficiencies of iron uptake and its subsequent release increased when the cells were exposed to increased concentrations of iron. Based on these observations, iron uptake/release appears to increase under conditions where DMT1/FPN1 is increased. Fourth, a decrease in iron uptake was observed in airway epithelial cells of Belgrade rats that are defective in DMT1 gene (Ghio AJ, unpublished results). Fifth, the apical membrane location of FPN1 as described above is consistent with FPN1's role in iron exporting in these cells. In the lung, airway epithelial lining serves as the first line of lung defense against exogenous invaders. Proteins and other products secreted by airway epithelial cells play important functions in detoxification and removal of harmful substance. We have shown that the airway epithelial cells are very tolerant to iron toxicity. These cells can efficiently take up a high amount of NTBI and subsequently release this metal as less-active protein-bound irons. A concentration-dependent linear uptake and release were observed between 0 and 1,000 µM of iron. Detectable cell damage caused by iron toxicity probably occurred only after the cells were exposed to >1,000 µM of the metal. In our experimental system, the recovery of a small amount of iron from the basolateral chamber during the subsequent release likely reflects a leakage of the monolayer culture cells exposed to a high concentration of iron. It is reasonable to speculate that cells that are not involved in iron absorption are more susceptible to iron toxicity. A recent study on neuronal cells demonstrated a >50% of cell death after the cells were exposed to iron for 2 days at a concentration as low as 40 µM (2). A subpopulation of these cells became resistant to iron with an adaptive response consisting of increased synthesis of FPN1 and ferritin and decreased synthesis of DMT1. In our study, upregulation of key iron transporters together with the augmentation of iron mobilization after exposure to iron in cells that do not function to meet the nutritional requirement of the living system can be explained if these activities are employed to reduced the oxidative stress and injury caused by the metal. This is consistent with our finding that NTBI was transported to the cell interior and then released as protein-bound iron. Interestingly, most of the newly acquired iron was released shortly following the uptake, and the original endogenous cellular iron was not involved in the release. This further supports our hypothesis that mobilization of the extracellular iron by these cells serves to detoxify catalytically active iron. Our proposed model of DMT1 and FPN1 in iron detoxification in the airway epithelia compared with their roles in iron absorption in the duodenal epithelia is presented in Fig. 8.

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Fig. 8. Comparison of the roles of divalent metal transporter-1 (DMT1) and FPN1 in iron metabolism in duodenal epithelia and airway epithelia. DMT1-mediated iron uptake across the apical membrane and FPN1-mediated iron export across the basolateral membrane into the circulation are responsible for nonheme nutritional iron absorption in the enterocytes. DMT1-mediated nontransferrin-bound iron uptake from airway lumen and FPN1-mediated export of iron across the apical membrane into the lumen as protein-bound complexes are 2 key components in iron detoxification by airway epithelial cells.
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Consistent with our proposed model for iron detoxification, we have identified ferritin and transferrin as major components of iron-bound complexes released by airway epithelial cells. These two iron-binding proteins are much more stable than free iron and seldom participate in redox reactions. The fate of the secreted protein-bound iron is not clear. However, a significant amount of the iron complexes may eventually be expelled (3). The mechanism(s) involved in converting NTBI to ferritin- and transferrin-bound iron for export remains unclear. Airway epithelial cells are capable of synthesizing transferrin (33) and ferritin (13). With exposure to iron, synthesis and secretion of ferritin by these cells were greatly increased (13). Increased release of ferritin and iron has also been demonstrated in iron-loaded alveolar macrophages from cigarette smokers (32). In isolated Kupffer cells, close to 50% of iron acquired from ingested red cells is released within 24 h in the form of ferritin. This release appeared to be associated with the ferritin-mediated transfer of iron from Kupffer cells to hepatocytes (26). In enterocytes, several proteins including FPN1, hephastin, transferrin receptor, and hemachromnatosis (Hfe) protein are believed to participate in a tightly controlled fashion, exporting iron across the basolateral membrane into the circulation as transferrin-bound iron. Although the role of each protein in this process is yet to be defined, we have found that airway epithelial cells are capable of synthesizing ceruloplasmin (34), a ferroxidase that is believed to be functionally equivalent to hephastin. In addition, hephastin mRNA has also been detected in the lung (29), although the specific lung cells that express this protein has not been delineated. More studies are needed to determine whether duodenal epithelial cells and airway epithelial cells share similar mechanisms for FPN1-mediated iron export even though the direction of export differs in these two cells. Mice deficient in genes coding for these proteins should be very useful in this endeavor.
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GRANTS
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This work was supported by National Institutes of Health Grants R01HL-68842 (to F. Yang) and R01DK-53079 (to D. J. Haile), a Veterans Affairs Administration Merit Grant Award (to D. J. Haile), and a Morrison Trust research grant (to F. Yang).
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ACKNOWLEDGMENTS
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The microscopic imaging work was conducted in the Digital Optical Imaging Facility at the University of Texas Health Science Center at San Antonio. The authors thank Drs. James Lechleiter and Victoria Frohlich for advice on the confocal microscopic imaging and Emily Van Beveren, Irma Oliva, Ngoc-Bich Nguyen, and Kimberly Marquess for technical assistance.
Disclaimer: This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for Publication. Approval does not signify that the contents necessarily reflect the views and polices of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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FOOTNOTES
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Address for reprint requests and other correspondence: F. Yang, Dept. of Cellular & Structural Biology, Univ. of Texas Health Science Center, San Antonio, TX 78229 (E-mail: yangf{at}uthscsa.edu); A. J. Ghio, National Health and Environmental Effects Research laboratory, Environmental Protection Agency, Research Triangle Park, NC 27711 (E-mail: ghio.andy{at}epa.gov)
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.
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REFERENCES
|
---|
- Abboud S and Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 275: 1990619912, 2000.[Abstract/Free Full Text]
- Aguirre P, Mena N, Tapia V, Arredondo M, and Nunez MT. Iron homeostasis in neuronal cells: a role for IREG1. BMC Nerosci 6: 3, 2005.[CrossRef]
- Alexis NE, Richards JH, Carter JD, and Ghio AJ. Iron-binding and storage proteins in sputum. Inhal Toxicol 14: 387400, 2002.[CrossRef][ISI][Medline]
- Baldwin SR, Grum CM, Boxer LA, Simon RH, Ketai LH, and Devall LJ. Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet 1: 1113, 1986.[ISI][Medline]
- Canonne-Hergaux F, Gruenheid S, Ponka P, and Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 93: 44064417, 1999.[Abstract/Free Full Text]
- Cochrane CG, Spragg R, and Reval SD. Pathogenesis of the adult respiratory distress syndrome: evidence of oxidant activity in bronchoalveolar lavage fluid. J Clin Invest 71: 754758, 1983.[ISI][Medline]
- Colton T. Statistics in Medicine. Boston, MA: Little, Brown, 1974.
- Devalia V, Carter K, Walker AP, Perkins SJ, Worwood M, May A, and Dooley JS. Autosomal dominant reticuloendothelial iron overload associated with a 3-base pair deletion in the ferroportin 1 gene (SLC11A3). Blood 100: 695697, 2002.[Abstract/Free Full Text]
- Donovan A, Brownliet A, Zhou Y, Shepard J, Pra SJ, Moynihan J, Paw BH, Drejer A, Barut B, Zapata A, Law TC, Brugnarall C, Lux SE, Pinkusl GS, Pinkusi JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, and Zon LI. Positional cloning of zebrafish ferroportin 1 identifies a conserved vertebrate iron exporter. Nature 403: 776780, 2000.[CrossRef][ISI][Medline]
- Edwards DK, Dyer WM, and Northway WH Jr. Twelve years experience with bronchopulmonary dysplasia. Pediatrics 59: 839846, 1977.[Abstract]
- Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, and Andrews NC. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for DMT1 in endosomal iron transport. Proc Natl Acad Sci USA 95: 11481153, 1998.[Abstract/Free Full Text]
- Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF, and Andrews NC. Microcytic anemia mice have a mutation in Nramp, a candidate iron transporter gene. Nat Genet 16: 383386, 1997.[CrossRef][ISI][Medline]
- Ghio AJ, Carter JD, Samet JM, Reed W, Quay J, Dailey LA, Richards JH, and Devlin R. Metal-dependent expression of ferritin and lactoferrin by respiratory epithelial cells. Am J Physiol Lung Cell Mol Physiol 274: L728L736, 1998.[Abstract/Free Full Text]
- Ghio AJ, Wang XS, Sibajoris R, Garrick MD, Piantadosi CA, and Yang F. DMT1 expression is increased in the lungs of hypotransferrinemic mice. Am J Physiol Lung Cell Mol Physiol 284: L938L944, 2003.[Abstract/Free Full Text]
- Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Cloning and characterization of a proton-coupled mammalian metal-ion transporter. Nature 388: 482488, 1997.[CrossRef][ISI][Medline]
- Ke Y, Reddel RR, Gerwin BI, Miyashita M, McMenamin M, Lechner JF, and Harris CC. Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation. Differentiation 38: 6066, 1988.[ISI][Medline]
- Kennedy TP, Dodson R, Rao NV, Ky H, Hopkins C, Baser M, Tolley E, and Hoidal JR. Dusts causing pneumoconiosis generate OH and produce hemolysis by acting as Fenton catalysis. Arch Biochem Biophys 269: 359364, 1989.[CrossRef][ISI][Medline]
- Ludwiczek S, Aigner E, Theurl I, and Weiss G. Cytokines-mediated regulation of iron transport in human monocytic cells. Blood 101: 41484154, 2003.[Abstract/Free Full Text]
- McKie AT and Barlow DJ. The SLC40 basolateral iron transporter family (IREG1/ferroportin/MTP1). Pflügers Arch 447: 810806, 2004.
- McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, Miret S, Bomford A, Peters TJ, Farzaneh F, Hediger MA, Hentze MW, and Simpson RJ. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 5: 299309, 2000.[CrossRef][ISI][Medline]
- Montosi G, Donovan A, Totaro A, Garuti C, Pignatti E, Cassanelli S, Trenor CC, Gasparini P, Andrews NC, and Pietrangelo A. Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 108: 619623, 2001.[Abstract/Free Full Text]
- Njajou OT, Vaessen N, Joosse M, Berghuis B, van Dongen JW, Breuning MH, Snijders PJ, Rutten WP, Sandkuijl LA, Oostra BA, van Duijn CM, and Heutink P. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat Genet 28: 213214, 2001.[CrossRef][ISI][Medline]
- Northway WH. Bronchopulmonary distress: then and now. Arch Dis Child 65: 10761081, 1990.[ISI][Medline]
- Pietrangelo A. The ferroportin diseases. Blood Cells Mol Dis 32: 131138, 2003.[CrossRef][ISI]
- Schapira RM, Ghio AJ, Effros RM, Morrisey J, Almagro UA, Dawson CA, and Hacker AD. Hydroxyl radical production and lung injury in the rat following silica or titanium dioxide instillation in vivo. Am J Respir Cell Mol Biol 12: 220226, 1995.[Abstract]
- Sibille JC, Kondo H, and Aisen P. Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. Hepatology 8: 296301, 1988.[ISI][Medline]
- Su MA, Trenor CC, Fleming JC, Fleming MD, and Andrews NC. The G185R mutation disrupts function of the iron transporter Nramp2. Blood 92: 21572163, 1998.[Abstract/Free Full Text]
- Turi JL, Jaspers I, Dailey LA, Madden MC, Brighton LE, Carter JD, Nozik-Graycki E, Piantadosi CA, and Ghio AJ. Oxidative stress activates anion exchange protein 2 and AP-1 in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 283: L791L798, 2002.[Abstract/Free Full Text]
- Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, and Anderson GJ. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 21: 195199, 1999.[CrossRef][ISI][Medline]
- Wang X, Ghio AJ, Yang F, Dolan KG, Garrick MD, and Piantadosi CA. Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 282: L987L995, 2002.[Abstract/Free Full Text]
- Wardrop SL and Richardson DR. Interferon- and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophages. Eur J Biochem 267: 65866593, 2000.[Abstract/Free Full Text]
- Wesselius LJ, Nelson ME, and Skikne BS. Increased release of ferritin and iron by iron-loaded alveolar macrophages in cigarette smokers. Am J Respir Crit Care Med 150: 690695, 1994.[Abstract]
- Yang F, Friedrichs WE, and Coalson JJ. Regulation of transferrin gene expression during lung development and injury. Am J Physiol Lung Cell Mol Physiol 273: L417L426, 1997.[Abstract/Free Full Text]
- Yang F, Friedrich WE, deGraffenried LA, Herbert DC, Weaker FJ, Bowman BH, and Coalson JJ. Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation. Am J Respir Cell Mol Biol 14: 161169, 1996.[Abstract]
- Yang F, Liu XB, Quinones M, Melby PC, Ghio AJ, and Haile DJ. Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation. J Biol Chem 277: 3978639791, 2002.[Abstract/Free Full Text]
- Yang F, Wang X, Haile DJ, Piantadosi CA, and Ghio AJ. Iron increases the expression of an iron export protein MTP1 in lung cells. Am J Physiol Lung Cell Mol Physiol 283: L932L939, 2002.[Abstract/Free Full Text]
- Yeh KY, Yeh M, Watkins JA, Rodriguez-Paris J, and Glass J. Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression. Am J Physiol Gastrointest Liver Physiol 279: G1070G1079, 2000.[Abstract/Free Full Text]
- Zoller H, Koch RO, Theurl I, Obrist P, Pietrangelo A, Montosi G, Haile DJ, Vogel W, and Weiss G. Expression of the duodenal iron transporters divalent -metal transporter 1 and ferroportin 1 in iron deficiency and iron overload. Gastroenterology 120: 14121419, 2001.[ISI][Medline]