The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe3+ concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.
- membrane transporters
iron is an essential micronutrient utilized in almost every aspect of normal cell function. The chemistry of iron presents difficulties in its acquisition by the cell. In aqueous solutions, Fe3+ forms oxyhydroxides, which generally are biologically inaccessible. As a result, cells have developed specific strategies to procure adequate iron for cellular function and homeostasis.
Natural resistance-associated macrophage proteins (Nramp) are a group of transporters in vertebrates that are representative of a small family of structurally and functionally related polypeptides conserved across numerous species, with homologs identified in yeasts, bacteria, worms, flies, and plants. This family of proteins is defined by the presence of a highly conserved hydrophobic core encoding 10 transmembrane domains, with additional similarity clustered in certain intracytoplasmic and extracytoplasmic loops (6). Evolutionary conservation suggests that a fundamental function may be common to all these proteins.
Nramp1 is a 90- to 100-kDa phosphoglycoprotein that confers resistance to intracellular pathogens through an uptake of divalent cations (29, 33). Such transport results in a deficiency in metal requisite for replication of the microbial. Nramp1 is expressed in the phagosomal membrane of neutrophils and macrophages and is inducible in the latter by exposure to cytokines and endotoxin (1, 7, 14).
Nramp2 has also been called divalent metal transporter-1 (DMT1) or divalent cation transporter-1 (DCT1) and is expressed in most tissues and cell types as an integral membrane protein (90–100 kDa) modified by glycosylation (32). Nramp1 and Nramp2/DMT1/DCT1 share 77% homology and have very similar (10 vs. 12 transmembrane) predicted secondary structures (6, 16). Nramp2/DMT1/DCT1 is localized primarily in recycling endosomes and at the plasma membrane colocalizing with transferrin. This protein also functions as a divalent metal cation transporter (17). Transport is coupled to proton movement, dependent on the cell membrane potential, and may include manganese, cobalt, nickel, zinc, copper, lead, and cadmium in addition to iron.
In the proximal duodenum, Fe3+ is reduced to Fe2+, which is then transported into the epithelial cell by Nramp2/DMT1/DCT1 (34). Microcytic anemic (mk) mice and Belgrade rats carry the same missense mutation in Nramp2/DMT1/DCT1 (9-11). This mutation, in which glycine-185 is changed to arginine (G185R), occurs within predicted transmembrane domain 4 of the protein (30). Subsequently, the mk mouse and the Belgrade rat demonstrate diminished intestinal iron absorption. The inherited defect of iron uptake in these naturally occurring animal mutants corroborated the hypothesis that Nramp2/DMT1/DCT1 is the transferrin-independent system responsible for dietary iron absorption in the intestine. The transient overexpression of the wild-type Nramp2/DMT1/DCT1 in HEK-293T cells stimulates cellular iron uptake, whereas that with the G185R mutation did not (15), further substantiating the participation of this protein in metal transport.
Nramp2 generates two alternatively spliced mRNAs that differ at their 3′-untranslated region by the presence or absence of an iron-response element (IRE). IREs are found in noncoding portions of certain proteins' mRNAs that are posttranscriptionally regulated in response to cellular iron levels (22, 23). The presence of an IRE suggested that Nramp2/DMT1/DCT1 levels may also be modulated by iron. However, decrements in Nramp2/DMT1/DCT1 protein after iron exposure predicted by the placement of the IRE at the 3′-untranslated region indicate that transport would decrease with increased concentrations. This cell response would be opposite that required to detoxify the oxidative stress presented by the metal in the lung. There also appears to be a potential for transcriptional control of Nramp2/DMT1/DCT1 exerted by iron. Nramp2/DMT1/DCT1 mRNA in Caco-2 cells is decreased by increasing cellular iron availability (17). However, an increase in Nramp2/DMT1/DCT1 protein staining and polarization to the sinusoidal membranes was noted in hepatocytes with iron excess (31). This modulation of mRNA levels was specific to iron. Consequently, regulation of Nramp2 expression appears to be tissue specific.
The capacity of Nramp2/DMT1/DCT1 to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent and -dependent uptake of iron in peripheral tissues. The lung is constantly exposed to iron. The upper respiratory tract is exposed daily to ∼10 μg of iron in an individual breathing with a tidal volume of 500 ml, a respiratory rate of 20/min, and a <10-μm particulate matter concentration of 50 μg/m3 containing 1.5% soluble iron. By promoting iron transport, Nramp2/DMT1/DCT1 could function in the lung to diminish the oxidative stress associated with the metal. We tested the hypothesis that iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal.
MATERIALS AND METHODS
All reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified.
Culture of BEAS-2B cells.
BEAS-2B cells, an immortalized line of normal human bronchial epithelium derived by transfection of primary cells with simian virus-40 early-region genes, were used in all studies. This particular subclone undergoes squamous differentiation in response to serum (21). Cells were grown to 90–100% confluence on uncoated plastic 12-well plates in ketinocyte growth medium (KGM; Clonetics), which is essentially MCDB 153 medium supplemented with 5 ng/ml human epidermal growth factor, 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.15 mM calcium, bovine pituitary extract, 0.1 mM ethanolamine, and 0.1 mM phosphoethanolamine. Previous investigation demonstrated that the response of BEAS-2B cells to iron is similar to that of normal human bronchial epithelial cells (12).
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 was ∼10 ppb.
Measurement of membrane-bound iron concentrations.
BEAS-2B cells were grown to 90–100% confluence on plastic 12-well plates in 1.0 ml of KGM. The medium was then switched to HEPES-buffered saline solution (HBSS), and the cells were exposed to ferric ammonium citrate (FAC) for 1 h. The supernatant was removed, and the cells were washed twice with HBSS. Surface-bound iron was then removed by incubating the cells with 1.0 ml of 140 mM NaCl, 10 mM PIPES, 5.0 mM sodium dithionite, and 5.0 mM bathophenanthrolene disulfonic acid (BPS) (13). The supernatant was removed and centrifuged at 600g for 10 min. Iron in the supernatants was measured using ICPAES.
Measurement of total cellular nonheme iron concentrations.
Epithelial cells were grown to 90–100% confluence on plastic 12-well plates in 1.0 ml of KGM. The medium was then switched to HBSS, and the cells were exposed to FAC for 1 h. HBSS with the metal was removed, and 1.0 ml of 3 N HCl containing 10% trichloroacetic acid was added. The cells were scraped into the 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 using ICPAES.
Assay for intracellular concentrations of iron.
BEAS-2B cells were grown to 90–100% confluence on plastic 12-well plates in 1.0 ml of KGM. Medium with 0.5 μM calcein-AM (Molecular Probes, Eugene, OR) was then added. After incubation for 1 h at 37°C, excess calcein-AM was removed, and the cells were washed twice with HBSS. FAC in HBSS was added to the BEAS-2B cells for 1 h. The HBSS with the metal was then removed, the cells were washed twice with HBSS, and the intensity of the fluorescence signal was measured with a luminescence spectrometer (model LS 50, Perkin-Elmer) with excitation at 486 nm and emission at 517 nm (4).
BEAS-2B cells were grown to confluence in 12-well plates (Costar, Cambridge, MA) and exposed to FAC for 24 h. The supernatant was removed. The cells were washed twice with PBS (Life Technologies, Grand Island, NY) and lysed with 4 M guanidine thiocyanate (Boehringer Mannheim, Indianapolis, IN), 50 mM sodium citrate, 0.5% sarkosyl, and 0.01 M dithiothreitol. After the cells were dislodged from wells with scrapers (Costar), lysates were sheared with four passes through a 22-gauge syringe. Total RNA (100 ng) was reverse transcribed (Maloney's murine leukemia virus reverse transcriptase; Life Technologies). Quantitative PCR was performed using TaqMan polymerase with detection of SYBR green fluorescence on a sequence detector (ABI Prism 7700, PE Biosystems, Foster City, CA). Nramp2 mRNA levels were normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Relative quantitation of Nramp2 and GAPDH mRNA was based on standard curves prepared from serially diluted mouse mast cell cDNA. The following sense and antisense sequences were employed: 5′-GGAGCAGTGGCTGGATTTAAGT-3′ (sense) and 5′-CCACTCCCAGTCTAGCTGCAA-3′ (antisense) for Nramp2/DMT1/DCT1 with IRE, 5′-TTTGTCGTCACTTTTCTTGAATTGTT-3′ (sense) and 5′-GGTTTCTGGATCTTGTTACTGGATATT-3′ (antisense) for Nramp2/DMT1/DCT1 without IRE, and 5′-GAAGGTGAAGGTCGGAGTC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense) for GAPDH. Nramp2/DMT1/DCT1 oligonucleotides were isoform specific.
Western blot analysis.
Cells were washed with ice-cold PBS, lysed with buffer containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitors (Cocktail Set III, Calbiochem, La Jolla, CA), and then sheared through a 22-gauge needle. Protein content was determined using the Bradford assay (Bio-Rad, Hercules, CA). The remainder of the sample was mixed with an equal volume of 4× sample loading buffer (0.5 M Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 0.7 mM β-mercaptoethanol, and 0.05% bromphenol blue).
Protein samples (50 μg) were separated by electrophoresis on a 4–15% SDS acrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 3% nonfat milk in PBS and incubated with an antibody directed against the Nramp2/DMT1/DCT1 with or without the IRE. Preparation of isoform-specific antibodies has been previously described (28). The membrane was stained with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) and developed using enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Tissue sections were cut, floated on a protein-free water bath, mounted on silane-treated slides (Fisher, Raleigh, NC), and air-dried overnight. The slides were heat fixed at 600°C in a slide dryer (Shandon Lipshaw, Pittsburgh, PA) for 10 min and cooled to room temperature. Sections were then deparaffinized and hydrated to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with H2O2 in absolute methanol (30% H2O2 in 30 ml of methanol) for 8 min. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in PBS. After treatment with Cyto Q Background Buster (Innovex Biosciences) for 10 min, slides were incubated with the primary antibody diluted 1:100 in 1% bovine serum albumin for 45 min at 37°C. Slides were incubated with biotinylated linking antibody from Stat-Q Staining System (Innovex Biosciences) for 10 min at room temperature and washed with PBS, and peroxidase enzyme label from Stat-Q Staining System was applied. After tissue sections were incubated for 10 min at room temperature and washed with PBS, they were developed with 3,3′-diaminobenzidine tetrahydrochloride for 3 min at room temperature. Sections were counterstained with hematoxylin, dehydrated through alcohols, and cleared in xylene, and coverslips were applied using a permanent mounting medium.
Values are means ± SE. Differences between multiple groups were compared using one-way analysis of variance (8) with Scheffé's post hoc test test. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.
Exposure of BEAS-2B cells to FAC in HBSS resulted in a decrease in the concentration of metal in the supernatant that was dependent on incubation time (Fig. 1 A). Similarly, the transport of iron into BEAS-2B cells in HBSS was concentration dependent (Fig. 1 B). Membranes will take up metals that bind reversibly to surface anionic sites on the cell. Membrane-associated iron was measured to establish that the metal was truly transported into the cell rather than adsorbed onto the membrane in increasing concentrations. After exposure to 0–100 μM FAC for 1 h, BEAS-2B cells had measurable concentrations of membrane-associated iron (Fig. 1 C). However, this capacity did not change with exposures to increasing concentrations of FAC. Subsequently, membrane-bound iron could not be responsible for increased cell concentrations of iron and diminished supernatant concentrations. In contrast, total nonheme iron in the BEAS-2B cells did increase with the concentration of FAC to which the cells were exposed (Fig. 1 D). To verify transport of the metal into the cell, BEAS-2B cells were incubated with the fluorescent probe calcein-AM for 1 h and then exposed to 0–200 μM FAC for 1 h. BEAS-2B cells incubated with the label (∼100 units) displayed a ∼20-fold increase in fluorescence relative to cells not exposed to calcein-AM (∼5 units). After FAC was included in the HBSS, the fluorescent signal diminished in proportion to initial iron concentration, consistent with an intracellular uptake of the metal (Fig. 1 E).
The mRNA isoform without an IRE increased after 24 h of exposure of BEAS-2B cells to FAC (Fig.2 A). This increase in mRNA was dependent on FAC concentration. In contrast, the mRNA isoform with an IRE did not demonstrate a significant change with exposure to FAC (Fig.2 B). By RT-PCR, there appeared to be considerably less mRNA with an IRE than without an IRE. Comparable to RT-PCR results, Western blot analyses supported a dose-dependent elevation in the expression of Nramp2 with an IRE (Fig. 3, Aand B), whereas the isoform with an IRE demonstrated no change (Fig. 3 C) after a 24-h exposure to iron.
Intratracheal exposure of rats to FAC resulted in an increased expression of Nramp2 without an IRE 24 h later (Fig.4, A and B). There was significantly less of the isoform with IRE than without IRE in the lungs exposed to saline (Fig. 4 C). In addition, there was little increase in uptake of the antibody to the Nramp2 with IRE after exposure to iron.
To determine whether iron transport could be enhanced, the effect of preexposure to iron and chelators on non-transferrin-bound iron (NTBI) transport was assessed in BEAS-2B cells. Pretreatment of the cells for 8 h with a variety of iron compounds significantly increased NTBI transport (Fig. 5 A). This stimulatory effect of metals on NTBI transport was not observed with incubations of shorter duration, suggesting upregulation of critical components of the iron transport system. Cell exposures to iron that was complexed to compounds that strongly coordinated the metal had no effect on iron uptake (i.e., ferroxamine and Fe-EDTA). Incubations of BEAS-2B cells with metal chelators (deferoxamine and EDTA at 0–1,000 μM for up to 8 h) before exposure to FAC had no significant effect on iron uptake. The experiment was repeated with pretreatment of BEAS-2B cells to HBSS or 100 μM ferric ammonium sulfate. After 8 h of incubation, the metal was removed, and FAC in HBSS was added (0–500 μM). There was an increase in the uptake of iron by the BEAS-2B cells after pretreatment with ferric ammonium sulfate (Fig. 5 B). Saturation was evident at the higher concentrations.
Finally, BEAS-2B cells were incubated with FAC along with other metal compounds to delineate the potential transport of metals other than iron by this same pathway involving Nramp2. The relationship between iron transport by BEAS-2B cells and the uptake of several other metals was investigated. The effects of vanadyl, nickel, and copper sulfates in the HBSS, at final concentrations of 500–1,000 μM, were most pronounced (Fig. 6). The ability of these metal salts to decrease iron uptake so dramatically suggested a common mechanism for metal transport. Similarly, the effects of cadmium, chromium, and zinc compounds on iron uptake by the BEAS-2B cells were highly significant (data not shown).
Molecular iron can be transported by a variety of cell types to an intracellular pool from a medium that contains no transferrin. Such transport has been demonstrated for microbes, plant cells, and selected animal cells (24, 25, 27, 35). Similar to the behavior of iron in these cells, the uptake of the metal by lung epithelial cells was time and concentration dependent. Although metal cations can bind reversibly to anionic sites onto cell membranes or other surfaces (e.g., glass and plastic), iron was not simply bound to the BEAS-2B cell membrane. Rather, fluorescence studies supported the hypothesis that the metal was transported to the cell interior.
mRNA for Nramp2/DMT1/DCT1 increased after BEAS-2B cell exposures to iron, but this was specific for the isoform without an IRE. Similarly, Western blot analysis demonstrated elevations in expression of Nramp2/DMT1/DCT1 without the IRE but not with the IRE. Finally, immunohistochemistry of the rat lung supported an upregulation of Nramp2/DMT1/DCT1 without an IRE after exposure to iron. This is in contrast to the response of intestinal cells to iron exposure, in which Nramp2/DMT1/DCT1 with an IRE increases with iron depletion as a result of posttranscriptional regulation by the IRE (17, 19). However, comparable to the results of this investigation, a transcriptional control of Nramp2/DMT1/DCT1 has been suggested in hepatocytes that increased expression of Nramp2/DMT1/DCT1 after iron exposure (31). In cells and tissues that do not function to meet the nutritional requirements of the living system, a transcriptional control of Nramp2/DMT1/DCT1 by iron to increase expression can be explained if this protein is employed to diminish the oxidative stress and injury this metal presents. Subsequently, it is possible that the regulation of expression of the protein can be modified in the lung and the liver to increase with metal exposure.
Pretreatment of BEAS-2B cells with iron-containing compounds increased NTBI transport, whereas chelator pretreatment had no effect. Metals can markedly stimulate their own rate of acquisition through an increase of NTBI transport (26). Such upregulation of iron transport has been considered to indicate the existence of a scavenger function for removal of NTBI (20). For example, cellular sequestration and detoxification of iron by ferritin require that the metal be transported across the membrane. With exposure to iron chelates, cells rapidly downregulate transferrin receptors (23), and, consequently, transferrin-dependent transport of iron across the membrane probably does not contribute to metal sequestration and the antioxidant function of ferritin. Thus an alternative mechanism of iron transport is required that can function to transfer the metal to intracellular ferritin after exposure to a chelate. NTBI transport can lead to incorporation of the metal, in part, into intracellular ferritin and, therefore, can play a role in the detoxification of iron (3).
Metals other than iron seem to compete for this same mechanism of cellular uptake. Iron uptake by BEAS-2B cells was diminished by the concomitant inclusion of high concentrations of other metal cations in the medium. These results suggest that iron is transported by a process that competes with other transition metal ions. This inhibition of iron uptake by other metal ions can be explained most simply by a common membrane carrier protein (5, 35), such as Nramp2/DMT1/DCT1.
Tissue damage resulting from an oxidative stress can ultimately be mediated by an increased availability of catalytically reactive iron (18). This metal can potentially contribute to the oxidative stress in the lower respiratory tract evident in several diseases. The lower respiratory tract is afforded antioxidant protection by a number of antioxidants, including glutathione, ceruloplasmin, vitamin E, and vitamin C. In addition to these antioxidants, the lower respiratory tract can contain the oxidative stress by binding the metal to specific proteins and, ultimately, storing it in a chemically less reactive form within ferritin (2). After disruption of iron homeostasis and elevation of available, catalytically active metal, an initial event in diminishing the metal-catalyzed oxidant generation would be an increased cellular uptake with storage within ferritin. It is possible that Nramp2/DMT1/DCT1 without an IRE provides the means for this transport of iron in the lower respiratory tract. Through the uptake of iron into a catalytically less reactive form (i.e., ferritin), Nramp2/DMT1/DCT1 could participate in diminishing oxidative stress in the lung.
We conclude that changes in iron transport by BEAS-2B cells after exposures to both metal compounds corresponded to differences in mRNA for Nramp2/DMT1/DCT1 without an IRE. Transcriptional regulation of Nramp2/DMT1/DCT1 by iron may contribute to its uptake and detoxification by these cells in the respiratory tract. It is also possible that Nramp2 participates in the inflammatory response to particles and fibers that are associated with an increase in concentrations of available iron. Whether this protein can meet the requirements of iron transport during such a challenge and, subsequently, diminish the oxidative stress is not known.
This report has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Address for reprint requests and other correspondence: A. J. Ghio, National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, NC 27711 (E-mail:).
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.
- Copyright © 2002 the American Physiological Society