1 Feist-Weiller Cancer Center and the Departments of 2 Medicine and 3 Molecular and Cell Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.
Caco-2 cells; intestinal iron transport
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INTRODUCTION |
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IRON TRANSPORT ACROSS the intestinal epithelium is complex and is usually thought of as occurring in three phases. In the first or uptake phase, Fe(III) at the brush-border membrane (BBM) is reduced by a ferrireductase (18) and then transported into the cell via the divalent metal transporter 1 (DMT1; also known as DCT1 and NRAMP2) (10, 14). In the second or transcellular phase, iron is transported across the cell to the basolateral surface. The transport may be via vesicular trafficking, as supported by our recent demonstration that DMT1 on the BBM of rat intestinal epithelium is internalized into vesicles when the rat is fed a bolus of iron (34). Alternatively, Fe(III) may be transported across the cell on chaperones such as the calreticulin-like protein mobilferrin (32). In the third phase, iron is transported out of the cell across the basolateral membrane. Recently, two proteins, ferroportin1 (also known as MTP and IREG1) (1, 7, 19) and hephaestin (33), have been hypothesized to be involved in basolateral membrane iron transport. Ferroportin1 is highly expressed in the duodenum and when transfected into Xenopus oocytes allows for the efflux of iron transported into the oocytes by DMT1. Hence, ferroportin1 is postulated as the ferrotransporter on the basolateral surface of intestinal epithelium (7). Hephaestin is a multicopper oxidase with homology to ceruloplasmin. In the sla mouse, a mutation in hephaestin impedes the transport of iron out of the enterocyte, suggesting that, although iron is transported into the cell as Fe(II), transport out of the cell requires oxidation to Fe(III) (33).
Iron homeostasis is regulated primarily at the level of intestinal iron uptake. With iron deficiency, iron transport into the epithelium is increased, as is the efficiency of iron transport from the intestinal into the systemic circulation. Conversely, iron overload decreases both the uptake into the mucosa and transport out of the intestine. The regulation of intestinal iron transport is controlled by the gene HFE, as evidenced by the increased iron transport that occurs with the C282Y mutation in HFE seen in hemochromatosis (8). The mechanism by which HFE regulates iron uptake is not yet certain (11, 16). HFE interacts with the transferrin receptor to modulate transferrin binding to the receptor (9). The mutated HFE found in hemochromatosis no longer binds to the transferrin receptor, and the cells functionally act as if iron deficient, although variable effects on DMT1 expression have been reported (11, 16). Recently, hepcidin, a liver-expressed antimicrobial peptide, has been implicated in regulation of iron transport out of the intestinal epithelium (12, 20).
The Caco-2 cell line grown in bicameral chambers has been used as a system to model intestinal iron uptake (2, 3, 5). These cells form a polarized monolayer when grown on a semiporous membrane, allowing demonstration of unidirectional iron transport from the apical to the basal chamber. Caco-2 cells have the components currently known to be required for iron transport, including DMT1, hephaestin, ferroportin1, and HFE. As in the intestine, iron deprivation of Caco-2 cells increases DMT1 expression. Iron transport is regulated similarly to that seen in the intestine, with iron transport being inversely proportional to the iron status of the cells. Although there is constitutive transport of iron across the Caco-2 cells, the transport of iron is stimulated by the presence of apo-transferrin in the basal chamber (3). Apo-transferrin can bind to the basolateral surface of the Caco-2 cells (23). By confocal microscopy, it is possible to demonstrate the endocytosis of apo-transferrin to a perinuclear location, in contrast to the basal compartment into which ferri-transferrin is transported (4).
Our laboratory has recently demonstrated (34) that on ingestion of iron DMT1 on the BBM of intestinal epithelium is internalized. That apo-transferrin is also internalized suggests that DMT1 and apo-transferrin may interact via transcytosis. Transcytosis in Caco-2 cells has been well described and in the context of iron transport could serve as a regulatory phenomenon, sequestrating DMT1 from the BBM and limiting iron uptake. Alternatively, or possibly additionally, transcytosis of DMT1 could provide a route by which iron may be transported across the cell. In this hypothesis, iron in vesicles internalized from the apical surface would interact with vesicles derived from the basal surface. The iron would be transferred to the basal surface-derived vesicles and then be transported out of the cell, presumably by ferroportin1. To substantiate that vesicles containing DMT1 do undergo transcytosis, we used confocal microscopy to demonstrate that DMT1 internalized from the BBM of Caco-2 cells colocalized in the same perinuclear compartment as basolateral-derived vesicles bearing apo-transferrin. Evidence of a pool of endosomes that contained both DMT1 and apo-transferrin was provided by isolation of endosomes from the Caco-2 cells by use of ultra-small paramagnetic particles (21, 26, 30). In the absence of iron in the apical chamber, the endosomes contained DMT1 as well as transferrin. The addition of iron to the apical chamber increased the DMT1 and transferrin content of the endosomes, with a marked increase in the amount of DMT1 relative to transferrin. Furthermore, when the apical chamber contained 59Fe and the basal chamber contained 125I-apo-transferrin, both moieties were found in endosomes containing the paramagnetic particles. Together, these observations suggest that, after internalization, DMT1 is involved in transcytosis and that this process, in cooperation with apo-transferrin, might provide a mechanism for iron passage from the apical to the basolateral membrane of Caco-2 cells.
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MATERIALS AND METHODS |
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Cell culture.
Caco-2 cells (HTB 37) from the American Type Culture Collection
(Rockville, MD) were maintained in DMEM supplemented with 10% FBS
(GIBCO, Gaithersburg, MD), 1% nonessential amino acids, and
antibiotics/antimycotic (100 U/ml penicillin-G, 100 U/ml
streptomycin, and 250 ng/ml Fungizone; GIBCO). Cells were grown in
6-mm-diameter Transwell bicameral chambers with 0.4-µm pore size
membranes (Costar, Cambridge, MA) coated with collagen. The collagen
film was applied to the filter as 50 µl of collagen solution (3 mg/ml, 60% ethanol, rat tail, type I; Boehringer-Mannheim, Mannheim,
Germany), and then the Transwells were inverted and dried under sterile
laminar airflow. Formation of a Caco-2 cell monolayer was monitored by measuring the transepithelial electrical resistance (TEER) with a
Millicell electrical resistance system (Millipore, Bedford, MA).
Confocal microscopy experiments were performed only after the TEER had
risen to a level indicating the formation of an intact monolayer (TEER
at least 250 · cm2) (2).
Typically, the cell monolayers were used after 12-14 days in
culture. The cell monolayers were depleted of serum proteins as
previously described (3). Before each experiment, cell
monolayers were washed with iron-free DMEM, without FBS, incubated for
20 min in DMEM three times, and transferred to a new well for at least
1 h before the start of the experiment.
Fluorochromes and transferrins. Texas red-labeled human transferrin and TO-PRO-3 were obtained from Molecular Probes (Eugene, OR). The labeled transferrin was reconstituted in water and rendered to the iron-free, apo form by lowering the solution pH to 4.5 with Na citrate in the presence of Chelex resin (30% vol/vol) under constant stirring. The pH of the solution was raised to 7.0 by dialysis against 0.15 M NaCl-10 mM HEPES (pH 7.0) in the presence of Chelex. The concentration of apo-transferrin was adjusted to 100 µM and made 100 µM in desferoxamine, frozen, and used within 6 mo. For experiments utilizing 125I-apo-transferrin, ferri-transferrin was labeled with 125I as previously described before preparation of apo-transferrin (17).
Laser scanning confocal microscopy and image analysis. The Caco-2 cell monolayers were observed under a laser scanning confocal microscope (Bio-Rad MRC 1024 scan head/Nikon Diaphot microscope). Images were collected utilizing a ×60 Nikon (apo-planar DIC) oil objective. Images for analysis were collected at a 512 × 512-pixel resolution. The Caco-2 cell monolayer was optically sectioned in the z-axis with a step size of 2 µm to give ~12 sections per imaged field. The images were analyzed by Lasersharp software (Bio-Rad), and pictures and graphs were generated in Adobe Photoshop and Excel (v. 6.0; Microsoft), respectively.
Antibodies to DMT1. The production of an antibody against the DMT1 protein has been described previously (34). In brief, we synthesized a polypeptide with amino acid sequences deduced from the iron-responsive element (IRE) isoform of rat DMT1 cDNA (14) consisting of amino acids 540 to 553 (CGRSVSISKVILSE) near the COOH terminus of the protein. The polypeptide was covalently linked to keyhole limpet hemocyanin, and the conjugate was used to inoculate New Zealand White rabbits. The antiserum, designated anti-540, has been previously shown to be specific to DMT1 (34).
Immunohistochemistry. Caco-2 cells grown to confluence on semiporous membranes were washed extensively with PBS for 15 min, fixed with 2% paraformaldehyde in PBS for 20 min at 4°C, and then permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were then washed extensively with PBS and blocked with 5% BSA and 1% goat serum in PBS for 1 h at room temperature. The cells were then incubated with anti-540 antibody or preimmune rabbit serum at a 1:500 dilution in PBS for 1 h at room temperature. Following extensive washing with PBS, the cell layers were incubated with Alexa 488-labeled goat anti-rabbit IgG (Molecular Probes) at a 1:500 dilution and with a 1:1,000 dilution of TO-PRO-3 for 1 h at room temperature. The cell monolayers were then washed extensively with PBS and mounted with ProLong Antifade kit (Molecular Probes).
Isolation of endosomes with superparamagnetic colloidal particles. Superparamagnetic colloidal iron particles coated with dextran with an average diameter of 8 nm were prepared by the procedure described by Rodriguez-Paris and colleagues (21, 26, 30). Caco-2 cells were incubated with the paramagnetic particles either in the basal or apical chamber for 20 min. The cells were washed extensively with Hanks' balanced saline-1 mM EDTA, scraped from the membrane, and disrupted by two passages in a stainless steel homogenizer (clearance 0.12 mm) in homogenization buffer [250 mM sucrose, 10 mM Tris (pH 7.4), 2 mM EDTA, 1 mM PMSF]. The homogenate was centrifuged at 1,000 g for 5 min, and the postnuclear supernatant was passed over a stainless steel wire mesh column surrounded by a 1.2-T magnet. The column was washed extensively, and endosomes containing the paramagnetic particles were collected by turning off the magnet, rinsing the column with homogenization buffer, and pelleting the endosomes at 45,000 g for 1 h at 4°C.
Statistical analysis. Where indicated, statistical comparisons were made by using the unpaired Student's t-test.
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RESULTS |
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Anti-540 detects DMT1 in Caco-2 cells.
The anti-540 antiserum is directed to the unique COOH terminus of the
IRE-containing isoform of rat DMT1. The antiserum has been shown by us
(34) to interact specifically with DMT1 from rat tissues.
Although the amino acid sequences in DMT1 are highly conserved among
species, the unique COOH-terminal amino acid homology between rat and
human is ~70%. It was important then to demonstrate that anti-540
did interact specifically with DMT1 from Caco-2 cells, a cell line
derived from human intestine. Figure 1
shows the Western blot of Caco-2 lysates compared with rat duodenal lysates separated by SDS-PAGE. In both the Caco-2 cells (lane 1) and the rat lysate (lane 2), anti-540 detected a
band of ~43 kDa that was eliminated by the presence of the immunizing
peptide (lanes 3 and 4).
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DMT1 is expressed on the BBM of Caco-2 cells and colocalizes with
Texas red apo-transferrin after the addition of ferrous ascorbate.
DMT1 has been demonstrated on the BBM of rat (14), mouse
(6), and human intestinal epithelium (31). To
demonstrate that DMT1 is on the BBM of Caco-2 cells, cells starved of
iron overnight were exposed to ferrous ascorbate in the apical chamber and Texas red apo-transferrin in the basal chamber. After 20 min, the
cells were washed extensively and the membrane holding the cell layer
was embedded in JB-4 embedding solution (Polyscience, Warrington, PA),
sectioned, and stained with anti-540 antisera. As seen by
immunofluorescence microscopy (Fig.
2A), DMT1 is clearly expressed
on the apical surface of the cells, with some vesicles apparent in the
upper portion of the cell. Texas red apo-transferrin can also be seen
to have been transported well into the cells. (Under the conditions of
immunofluorescence microscopy, the TO-PRO-3-stained nuclei also appear
red, giving the red "block" appearance in some cells).
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Does the internalization of DMT1 result in increased DMT1
catabolism?
The apparent shift of DMT1 back toward the apical portion of the cell
seen in Fig. 3A could result from DMT1 undergoing exocytosis and returning to the BBM. Alternatively, with internalization DMT1
might undergo degradation and be replaced by newly synthesized DMT1
targeted to the apical surface. To distinguish between these two
possibilities, the synthetic and degradation rates of DMT1 were
analyzed. After overnight incubation without iron in the apical
chamber, the Caco-2 cells were incubated with
[35S]methionine in methionine-free medium in the apical
chamber in the presence or absence of apical iron. Cells were lysed at
various times up to 3 h, DMT1 was isolated by immunoprecipitation
with anti-540, and the [35S]methionine radioactivity was
determined by autoradiography of SDS-PAGE separation of the
immunoprecipitated DMT1 (Fig.
4A). The relative rates of
synthesis in arbitrary units from scanned autoradiographs were
223.7 ± 47.6 and 216.9 ± 28.2 for cells exposed or not
exposed to apical iron, respectively (means ± SE of 3 experiments). The failure of a bolus of iron to stimulate synthesis of
DMT1 in Caco-2 cells has been demonstrated previously
(27).
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The colocalization of DMT1 and apo-transferrin occurs for a finite
period of time and is specific for apo-transferrin.
To examine the length of time that DMT1 and apo-transferrin remained
colocalized, the two entities were visualized by following a cohort of
Texas red apo-transferrin into and out of the cells. In these
experiments, the Caco-2 cells were exposed to Texas red apo-transferrin
for 20 min and then the basal chamber buffer was replaced with buffer
containing nonfluorescent apo-transferrin. With these experiments, it
was also possible to discern whether apo-transferrin and DMT1
separated, with apo-transferrin returning to a basal compartment. In
Fig. 5, the optical sections along the
z-axis were reconstructed, allowing the cell layer to be
seen from a lateral view. After 20 min of exposure to Texas red
apo-transferrin, there was considerable colocalization of
apo-transferrin and DMT1. The colocalization persisted for 20 min of
chase. No colocalization and only small amounts of Texas red
apo-transferrin were observed at 40 min. By 60 min, no further Texas
red label could be seen in the cells. In these experiments,
apo-transferrin was never seen in the most basal sections of the cells,
suggesting that the apo-transferrin was exiting the cells at the
lateral, pericellular space. To examine the specificity of
colocalization, Caco-2 cells were incubated with Texas red
ferri-transferrin (Fig. 6B)
and FITC-labeled dextran (molecular weight 70,000; Fig. 6A).
In the latter case, DMT1 was detected using Alexa-594-labeled
goat anti-rabbit antiserum. In Fig. 6B the optical sections
along the z-axis were reconstructed for the combination of
DMT1 and ferri-transferrin 20 min after the addition of iron to the
apical chamber and ferri-transferrin to the basal chamber.
Ferri-transferrin remained in the basal portions of the cells, and no
colocalization was observed with DMT1 at 20 min, nor was colocalization
observed with longer incubation times (data not shown). Figure
6A is the reconstruction 20 min after the addition of iron
to the apical chamber and FITC-dextran to the basal chamber. Although
FITC-dextran migrated to a perinuclear region, only very slight
colocalization was seen with DMT1. These experiments support that Texas
red apo-transferrin is marking a unique population of vesicles that
interact with vesicles derived from the apical surface and that contain
DMT1.
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DMT1 and iron are present in endosomes derived from the basolateral
membrane.
To further demonstrate that DMT1 undergoes endocytosis and that the
endosomes carrying DMT1 interact with endosomes derived from the
basolateral membrane, we made use of the ability to separate endosomes
with ultra-small paramagnetic colloidal particles. In these
experiments, the ultra-small particles are trapped within the endosomal
space and serve as a marker for the endosomes, as previously described
both in Dictyostelium (21, 26) and mammalian cells (30). In the first set of experiments (Fig.
7A), endosomes were isolated
from Caco-2 cells that had been incubated without apical iron and with
(lane 3) or without (lanes 1 and 2)
apo-transferrin and with magnetic particles added either to the basal
chamber (lanes 1 and 3) or the apical chamber
(lane 2). The isolated endosomes were subjected to SDS-PAGE
and transferred to nylon membranes, and DMT1 and transferrin were
detected by Western blotting. Protein concentrations in all three lanes
were similar, as judged by staining the membranes with Ponceau red
before Western blotting. Similar amounts of DMT1 were present whether
magnetic beads were placed in the basal (lane 1) or apical
(lane 2) chamber. Since by confocal microscopy we never
observe endocytosis of apical DMT1 unless iron is present in the apical
chamber, these findings suggest that there is constitutive endocytosis
of the small amounts of DMT1 present on the basolateral surface of the
cells and that vesicles containing this DMT1 meet with endosomes from
the apical surface. When apo-transferrin was added to the basal chamber
(lane 3) and magnetic beads were in the apical chamber, the
isolated endosomes contained apo-transferrin and amounts of DMT1
similar to that observed in lanes 1 and 2. These
experiments support the observation of constitutive endocytosis of
apo-transferrin and the interaction of these endosomes with
apical-derived endosomes, as identified by the endocytosis of beads
from the apical chamber.
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The affect of apical Fe(II) on internalization of apo-transferrin. The internalization of DMT1 from the BBM of Caco-2 cells clearly required the presence of apical iron, as was the case in rat duodenum (34). It was of interest to determine if the presence of apical iron affected the internalization of apo-transferrin and conversely if the presence of apo-transferrin in the basal compartment affected the internalization of DMT1. The internalization of Texas red apo-transferrin and DMT1 was quantified in multiple experiments by optically sectioning Caco-2 cells by confocal microscopy. The number of pixels representing Texas red apo-transferrin in the section with the greatest amount of apo-transferrin in the presence of Fe(II) was 272 ± 63 and in the absence of Fe(II) was 195 ± 46 (means ± SE of 7 experiments; P < 0.05). In the converse experiment, the presence of apo-transferrin in the basal chamber had no effect on the internalization of DMT1 in response to iron.
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DISCUSSION |
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The transport of iron across the intestinal epithelium can be thought of as proceeding through three phases: transport across the BBM, intracellular transport from the apical portion of the cell to the basolateral compartment, and transport across the basolateral membrane and into the systemic circulation. In addition, the process of iron transport is regulated to allow for maintenance of iron homeostasis. A number of the components necessary for iron transport and regulation of the transport have been defined, especially those required for the uptake of iron into the cell and transport of the iron out of the cell. In the first phase, a ferrireductase located on the BBM is required (18, 22, 25). Then DMT1 located on the brush border of the intestinal epithelium transports Fe(II) into the cell (14, 31). For transport of iron out of the cell, ferroportin1 (also known as IREG1 and MTP) (1, 7, 19) and hephaestin (33) have been implicated. How iron is transported from the apical transporter to the basolateral transporter is as yet unknown. Separate pathways have been postulated for ferric and ferrous iron (32), and chaperones have been proposed to transport iron across the cell (32). The data presented in this report support a model of vesicular transport of the apical transporter DMT1 that may involve transcytosis with apical-derived vesicles interacting with basolateral-derived vesicles.
DMT1 has been identified on the BBM of intestinal epithelium (6, 30, 34). In mouse intestine, DMT1 was visualized in the brush border but also with a punctate appearance in the apical half of the cells, suggesting that DMT1 was present in vesicles (6). In other cell types, DMT1 has been identified both in early and late endosomes (13, 29, 31). In cells, such as erythroid precursors, in which ferri-transferrin is endocytosed into the cells bound to the transferrin receptor, it is not surprising that DMT1 would be located in endosomes because it is only after endocytosis of the transferrin-transferrin receptor complex that iron transport occurs. In K-562 cells, DMT1 is found in late endosomes or lysosomes, colocalizing with lysosomal-associated membrane protein 1 but not with transferrin receptor (29). This observation suggests that endosomes undergo fission, with vesicles containing the transferrin-transferrin receptor complex recycling to the membrane while a portion of the vesicles carrying iron and DMT1 fuse with lysosomes.
In intestinal epithelium, the question arises as to why DMT1 should undergo endocytosis. Is the internalization of DMT1 required for the transport of iron into the intestinal mucosa? That is, are iron and DMT1 located in the same internalized vesicle? Or does the internalization of DMT1 in some way regulate the uptake of iron? A regulatory role is suggested by the behavior of another metal transporter, the Menkes protein (ATP7A; MNK) that is involved in copper transport (24). The protein is normally found in the trans-Golgi network. Under elevated copper conditions, the Menkes protein is transported to the plasma membrane via clathrin-coated endosomes. During the exocytosis, the protein pumps copper from the cytosol into the vesicles (24). However, in Caco-2 cells the uptake of iron is linear for at least 3 h (Refs. 2 and 3 and unpublished studies), indicating that the internalization of DMT1 does not acutely decrease iron accumulation. In addition, in the current studies within the range of iron concentrations tested (0.1-10 µM), there was no difference in the extent of DMT1 internalized (data not shown). Hence, it seems more likely that DMT1 is involved in the vesicular trafficking of iron. Perhaps the internalization directs iron in apical vesicles to the components of the basolateral membrane required for iron transport of the cell.
It was with this reasoning that, having observed the vesicular uptake of DMT1 into Caco-2 cells, we examined whether DMT1 colocalized with vesicles derived from the basolateral surface. We used two markers for vesicles derived from the basolateral surface: apo-transferrin and ultra-small paramagnetic colloidal particles. We have previously shown that, when Caco-2 cells were grown in bicameral chambers, apo-transferrin offered from the basal chamber increased the constitutive transport of iron by increasing the efficiency of transport of iron out of the cell (3). Also, by confocal microscopy apo-transferrin could be visualized reaching a perinuclear compartment in the apical portion of the cells, whereas internalized ferri-transferrin remained in a more basal compartment (4). In the current studies, we show that DMT1 and apo-transferrin colocalize and then separate with the apo-transferrin signal decreasing, suggesting its exit from the cells. The colocalization was specific, because no colocalization was observed with ferri-transferrin, which remained in the basal portion of the cells. Also, FITC-labeled dextran, which was internalized to the perinuclear region (as well as to the more basal portions of the cells), did not colocalize with DMT1. These studies illustrate that there is specificity to vesicle trafficking and subsets of vesicles. Those whose intravesicluar space is marked with FITC-dextran distribute throughout the cell but do not colocalize with apical-derived vesicles carrying DMT1 in the membrane. Likewise, those vesicles with ferri-transferrin bound to the transferrin receptor have a different behavior than those bound with apo-transferrin. These results also support the observation of Nunez et al. (23) that, in contrast to other cell types, apo-transferrin has significant interactions with Caco-2 cells.
The paramagnetic particles offered in the basal chamber also served as a marker for vesicles derived from the basolateral membrane. In the absence of iron in the apical chamber, DMT1 was present in the endosomes isolated by the magnetic column with a ratio of DMT1 to transferrin significantly less than when iron was present. Both other laboratories (6) and ours (34) have shown that some DMT1 is present in the basolateral membrane and therefore might very well be present in vesicles undergoing endocytosis from the basolateral surface. With the addition of iron to the apical chamber, a maneuver that increases DMT1 endocytosis from the apical surface, the amount of DMT1 increased markedly relative to transferrin in vesicles isolated with the simultaneous exposure of the cells to magnetic particles in the basal chamber. This finding is consistent with apical-derived vesicles containing DMT1 fusing with basolateral-derived vesicles, which contained the magnetic particles. That the same relative amounts of DMT1 and transferrin were in endosomes isolated after the addition of paramagnetic particles to the apical chamber is again consistent with apical-derived endosomes containing DMT1 fusing with basolateral-derived vesicles containing transferrin. Furthermore, that the endosomes contained roughly equivalent amounts of 59Fe and 125I-apo-transferrin suggest that vesicles that contain DMT1 and transferrin also contain iron. Together, the colocalization of apo-transferrin with DMT1 observed by confocal microscopy and the results of vesicle isolation by paramagnetic beads suggest that at least some of the BBM DMT1 fuses with vesicles from the basolateral surface, as identified by containing either apo-transferrin or ultra-small paramagnetic particles. Further studies involving vesicle isolation will be required to quantify the fraction of iron that is transported across the Caco-2 cells by transcytosis.
The process of transcytosis involving transferrin has been previously described as occurring in Caco-2 cells (15). In these studies, apical uptake either of a particulate or a soluble marker could be demonstrated to interact with endocytic vesicles derived from the basal surface and containing transferrin. Mixing of the apical and basal labels took place in the apical portion of the cells. That DMT1 and apo-transferrin colocalize raises many questions. What cytosolic proteins mediate the endocytosis of DMT1 and the interaction of apical-derived vesicles with basal-derived vesicles containing apo-transferrin? How is iron passed from apical vesicles to basal vesicles? Are there roles for ferroportin1 and hephaestin in the process of transcytosis? One can hypothesize that ferroportin1, with a structure of multiple transmembrane domains, may serve both as a dock for vesicles containing iron and to transport iron out of the cells from the vesicles. Hephaestin as an oxidase may reside in the basal vesicles and be required for oxidation of iron, either to allow binding to internalized apo-transferrin or for export from the cell in the absence of apo-transferrin.
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ACKNOWLEDGEMENTS |
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This work was supported by grant DK-41279 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Feist-Weiller Cancer Center.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Glass, Feist-Weiller Cancer Center, 1501 Kings Highway, Shreveport, LA 71130 (E-mail: jglass{at}lsuhsc.edu).
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.
June 12, 2002;10.1152/ajpgi.00005.2002
Received 7 January 2002; accepted in final form 5 June 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abboud, S,
and
Haile DJ.
A novel mammalian iron-regulated protein involved in intracellular iron metabolism.
J Biol Chem
275:
19906-19912,
2000
2.
Alvarez-Hernandez, X,
Nichols GM,
and
Glass J.
Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers.
Biochim Biophys Acta
1070:
205-208,
1991[ISI][Medline].
3.
Alvarez-Hernandez, X,
Smith M,
and
Glass J.
The effect of apotransferrin on iron release from Caco-2 cells, an intestinal epithelial cell line.
Blood
91:
3974-3979,
1998
4.
Alvarez-Hernandez, X,
Smith M,
and
Glass J.
Apo-transferrin is internalized and routed differently from Fe-transferrin by Caco-2 cells: a confocal microscopy study of vesicular transport in intestinal cells.
Blood
95:
721-723,
2000
5.
Arredondo, M,
Orellana A,
Garate MA,
and
Nunez MT.
Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells.
Am J Physiol Gastrointest Liver Physiol
273:
G275-G280,
1997
6.
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:
4406-4417,
1999
7.
Donovan, A,
Brownlie A,
Zhou Y,
Shepard J,
Pratt SJ,
Moynihan J,
Paw BH,
Drejer A,
Barut B,
Zapata A,
Law TC,
Brugnara C,
Lux SE,
Pinkus GS,
Pinkus JL,
Kingsley PD,
Palis J,
Fleming MD,
Andrews NC,
and
Zon LI.
Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.
Nature
403:
776-781,
2000[ISI][Medline].
8.
Feder, JN,
Gnirke A,
Thomas W,
Tsuchihashi Z,
Ruddy DA,
Basava A,
Dormishian F,
Domingo R, Jr,
Ellis MC,
Fullan A,
Hinton LM,
Jones NL,
Kimmel BE,
Kronmal GS,
Lauer P,
Lee VK,
Loeb DB,
Mapa FA,
McClelland E,
Meyer NC,
Mintier GA,
Moeller N,
Moore T,
Morikang E,
Prass CE,
Quintana L,
Starnes SM,
Schatzman RC,
Brunke KJ,
Drayna DT,
Risch NJ,
Bacon BR,
and
Wolff RK.
A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.
Nat Genet
13:
399-408,
1996[ISI][Medline].
9.
Feder, JN,
Penny DM,
Irrinki A,
Lee VK,
Lebron JA,
Watson N,
Tsuchihashi Z,
Sigal E,
Bjorkman PJ,
and
Schatzman RC.
The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.
Proc Natl Acad Sci USA
95:
1472-1477,
1998
10.
Fleming, MD,
Trenor CC,
Su MA,
Foernzler D,
Beier DR,
Dietrich WF,
and
Andrews NC.
Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene.
Nat Genet
16:
383-386,
1997[ISI][Medline].
11.
Fleming, RE,
Migas MC,
Zhou X,
Jiang J,
Britton RS,
Brunt EM,
Tomatsu S,
Waheed A,
Bacon BR,
and
Sly WS.
Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.
Proc Natl Acad Sci USA
96:
3143-3148,
1999
12.
Fleming, RE,
and
Sly WS.
Hepcidin: a putative iron-regulatory hormone relevant to hereditary hemochromatosis and the anemia of chronic disease.
Proc Natl Acad Sci USA
98:
8160-8162,
2001
13.
Gruenheid, S,
Canonne-Hergaux F,
Gauthier S,
Hackam DJ,
Grinstein S,
and
Gros P.
The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.
J Exp Med
189:
831-841,
1999
14.
Gunshin, H,
Mackenzie B,
Berger UV,
Gunshin Y,
Romero MF,
Boron WF,
Nussberger S,
Gollan JL,
and
Hediger MA.
Cloning and characterization of a mammalian proton-coupled metal-ion transporter.
Nature
388:
482-488,
1997[ISI][Medline].
15.
Hughson, EJ,
and
Hopkins CR.
Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces.
J Cell Biol
110:
337-348,
1990[Abstract].
16.
Levy, JE,
Montross LK,
and
Andrews NC.
Genes that modify the hemochromatosis phenotype in mice.
J Clin Invest
105:
1209-1216,
2000
17.
Martinez-Medellin, J,
and
Schulman HM.
The kinetics of iron and transferrin incorporation into rabbit erythroid cells and the nature of stromal-bound iron.
Biochim Biophys Acta
264:
272-274,
1972[ISI][Medline].
18.
McKie, AT,
Barrow D,
Latunde-Dada GO,
Rolfs A,
Sager G,
Mudaly E,
Mudaly M,
Richardson C,
Barlow D,
Bomford A,
Peters TJ,
Raja KB,
Shirali S,
Hediger MA,
Farzaneh F,
and
Simpson RJ.
An iron-regulated ferric reductase associated with the absorption of dietary iron.
Science
291:
1755-1759,
2001
19.
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:
299-309,
2000[ISI][Medline].
20.
Nicolas, G,
Bennoun M,
Devaux I,
Beaumont C,
Grandchamp B,
Kahn A,
and
Vaulont S.
Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.
Proc Natl Acad Sci USA
98:
8780-8785,
2001
21.
Nolta, K,
Rodriguez-Paris JM,
and
Steck TL.
Analysis of successive endocytic compartment isolated from Dictyostelium discoideum by magnetic fractionation.
Biochim Biophys Acta
1124:
237-246,
1994.
22.
Nunez, MT,
Alvarez X,
Smith M,
Tapia V,
and
Glass J.
Role of redox systems on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells.
Am J Physiol Cell Physiol
267:
C1582-C1588,
1994
23.
Nunez, MT,
Nunez-Millacura C,
Beltran M,
Tapia V,
and
Alvarez-Hernandez X.
Apotransferrin and holotransferrin undergo different endocytic cycles in intestinal epithelia (Caco-2) cells.
J Biol Chem
272:
19425-19428,
1997
24.
Petris, MJ,
and
Mercer J.
The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal.
Hum Mol Genet
8:
2107-2115,
1999
25.
Raja, KB,
Simpson RJ,
and
Peters TJ.
Investigation of a role for reduction in ferric iron uptake by mouse duodenum.
Biochim Biophys Acta
1135:
141-146,
1992[ISI][Medline].
26.
Rodriguez-Paris, JM,
Nolta KV,
and
Steck LS.
Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation.
J Biol Chem
268:
9110-9116,
1993
27.
Sharp, P,
Tandy S,
Yamaji S,
Tennant J,
Williams M,
and
Srai SKS
Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.
FEBS Lett
510:
71-76,
2002[ISI][Medline].
28.
Su, MA,
Trenor CC,
Fleming JC,
Fleming MD,
and
Andrews NC.
The G185R mutation disrupts functions of the iron transporter Nramp2.
Blood
92:
2157-2163,
1998
29.
Tabuchi, M,
Yoshimori T,
Yamaguchi K,
Yoshida T,
and
Kishi F.
Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEP-2 cells.
J Biol Chem
275:
22220-22228,
2000
30.
Tan, LJ,
Ceman S,
Chervonsky A,
Rodriguez-Paris JM,
Steck TL,
and
Sant AJ.
Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II chain.
Eur J Immunol
27:
1479-1488,
1997[ISI][Medline].
31.
Tandy, S,
Williams M,
Leggett A,
Lopez-Jimenez M,
Dedes M,
Ramesh B,
Srai SK,
and
Sharp P.
Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells.
J Biol Chem
275:
1023-1029,
2000
32.
Umbreit, JN,
Conrad ME,
Moore EG,
and
Latour LF.
Iron absorption and cellular transport: the mobilferrin/paraferritin paradigm.
Semin Hematol
35:
13-26,
1998[ISI][Medline].
33.
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:
195-199,
1999[ISI][Medline].
34.
Yeh, KY,
Yeh M,
Watkins JA,
Rodriguez-Paris J,
and
Glass J.
Dietary iron induces rapid changes in rat intestinal divalent metal transporter (DMT1) expression.
Am J Physiol Gastrointest Liver Physiol
279:
G1070-G1079,
2000