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Am J Physiol Gastrointest Liver Physiol 290: G301-G309, 2006. First published September 22, 2005; doi:10.1152/ajpgi.00029.2005
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MUCOSAL BIOLOGY

Vesicular transport and apotransferrin in intestinal iron absorption, as shown in the Caco-2 cell model

Mizue Moriya and Maria C. Linder

Department of Chemistry and Biochemistry And Institute for Molecular Biology and Nutrition, California State University, Fullerton, California

Submitted 24 January 2005 ; accepted in final form 15 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential roles of vesicular transport and apotransferrin (entering from the blood) in intestinal Fe absorption were investigated using Caco-2 cell monolayers with tight junctions in bicameral chambers as a model. As shown previously, addition of 39 µM apotransferrin (apoTf) to the basolateral fluid during absorption studies markedly stimulated overall transport of 1 µM 59Fe from the apical to the basal chamber and stimulated its basolateral release from prelabeled cells, implicating endo- and exocytosis. Rates of transport more than doubled. Uptake was also stimulated, but only 20%. Specific inhibitors of aspects of vesicular trafficking were applied to determine their potential effects on uptake, retention, and basolateral (overall) transport of 59Fe. Nocodazole and 5'-(4-fluorosulfonylbenzoyl)-adenosine each reduced uptake and basolateral transport up to 50%. Brefeldin A inhibited about 10%. Tyrphostin A8 (AG10) reduced uptake 35% but markedly stimulated basolateral efflux, particularly that dependent on apoTf. Cooling of cells to 4°C (which causes depolymerization of microtubules and lowers energy availability) profoundly inhibited uptake and basolateral transfer of Fe (7- to 12-fold). Apical efflux (which was substantial) was not temperature affected. Our results support the involvement of apoTf cycling in intestinal Fe absorption and indicate that as much as half of the iron uses apoTf and non-apoTf-dependent vesicular pathways to cross the basolateral membrane and brush border of enterocytes.

endocytosis; exocytosis; polarized Caco-2 cell monolayers

MUCH IS KNOWN ABOUT INTESTINAL iron absorption, but many details remain to be discovered. Both Fe(II) and Fe(III) are absorbed from the diet, although uptake of Fe(II) predominates (6, 11–13, 26, 39). The capacity for absorption increases in iron deficiency and decreases when levels of body iron are high (12, 13, 26). Uptake of Fe(II) occurs through divalent metal transporter 1 (DMT1/ DCT1/Nramp2) (14, 17), located in the apical brush border (18, 29, 41), but also in apical and perinuclear endosomes (22, 29, 48). Fe(III) must first be reduced by a ferrireductase (Dcytb), also in the apical membrane (14), and is then guided to either DMT1 or another as yet unknown transporter (10, 11). For exit of iron at the other end of the enterocyte, there is ferroportin/IREG1, located in the basolateral membrane (1, 14, 31) but also found in endocytic vesicles and at the brush border (30, 42). How iron is delivered to ferroportin and/or crosses the cytoplasm remains a mystery, although unknown cytoplasmic iron-containing components (25, 26) and a pool of labile iron (24) have been detected. The cellular location and changes in expression of DMT1 and ferroportin in response to iron deficiency and excess are consistent with these transporters playing major roles in iron absorption, although DMT1 is also used by some other metal ions (7, 16, 18, 35, 38, 47). On the basis of whole organism studies, transfer of iron across the basolateral membrane is regulated more stringently than uptake across the brush border, the former responding to the iron needs of the body, the latter only to enterocyte iron status. Thus there tends to be a reciprocal relationship between cellular retention of dietary iron (and intracellular ferritin) and its transfer to the blood (25, 26, 32). This observation led to the early "mucosal block" theory, in which ferritin concentrations were viewed as regulating overall iron absorption by acting as a sink for iron and reducing its basolateral transfer (17, 26, 32).

In addition to a potential cytoplasmic route for iron across the enterocyte, there is evidence of vesicular transport or transcytosis. In this regard, the studies of Glass, Nunez, and colleagues with apotransferrin (apoTf) are of particular interest. With the use of polarized Caco-2 cell monolayers with tight junctions as a model, these investigators demonstrated that addition of apoTf to the "blood side" stimulated overall iron absorption in a dose-dependent manner (2, 4, 34, 50). Unlike ferric-transferrin (Tf; which also enters Caco-2 cells) (2, 3, 30, 33), they showed that apoTf traveled in endosomes to the apical region of the cell, where it colocalized with vesicles containing DMT1 (29, 30). It then gradually left that area, presumably exiting back into the basal fluid (29). These findings explain the detection of Tf (with or without iron) in mucosal extracts, the levels of which increased in iron deficiency (26, 32). They suggest a vesicular pathway involving iron entering endosomes at the brush border (perhaps bound to DMT1), binding to apoTf that has entered by endocytosis from the blood plasma at the basolateral membrane, and then being exocytosed (as iron Tf) back into the blood. In such a scenario, hephaestin, the copper-containing ceruloplasmin homolog that also has ferroxidase activity (5, 9, 40), might be in the same endosomes and serve to oxidize the incoming Fe(II) so it can bind to the apoTf [which only binds Fe(III)].

The studies reported here were designed to further evaluate the importance of vesicular transport to intestinal iron absorption, using the Caco-2 cell intestinal model where conditions can be carefully controlled and manipulated. Our results strongly support a major involvement of vesicular trafficking in overall transfer of iron from the intestinal lumen to the blood and across the enterocyte monolayer, in transport pathways that are dependent as well as not dependent on apoTf.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. DMEM was from Sigma (St. Louis, MO), and all supplemental reagents for the cell culture medium were from Cambrex Bio Science (Walkersville, MD). Twelve-well Transwell plates (3.0-µm pore size and 0.33-cm2 growth area) and rat tail collagen were from Costar (Cambridge, MA) and Roche (Indianapolis, IN), respectively. 59FeCl3 was from Perkin-Elmer (Wellesley, MA). Desferroxamine (DFO), brefeldin A (BFA), nocodazole, and 5'-(4-fluorosulfonylbenzoyl)adenosine hydrochloride (FSBA) were from Sigma. Tyrophostin A8 (AG10) and apoTf were from Calbiochem/EMD Bioscience (San Diego, CA).

Cells and cell culturing. Caco-2 cells, obtained from the American Type Culture Collection (Manassas, VA), were cultured and grown into monolayers, as described previously (50), in DMEM supplemented with 10 or 20% FBS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 1% antimycotic-antibiotic reagent (10,000 U/ml penicillin G, 25 µg/ml of amphotericin B, and 10,000 µg/ml of streptomycin). At 70–80% confluency, cells (50,000/well) were seeded on 12-well Transwell plates precoated with 50 µl of rat tail collagen. For transport studies, monolayers with tight junctions were used after reaching a resistance of 1,200–1,400 {Omega}.

In some cases, monolayers were pretreated with inhibitors of different aspects of vesicular transport for up to 2 h. Stocks of these inhibitors were prepared by dissolving them in DMSO and were diluted to desired concentrations with appropriate incubation fluids: DMEM in the case of AG10; DMEM with 1% FBS for nocodazole; HEPES buffer (in mM: 50 HEPES, 130 NaCl, 1 CaCl2, 1 MgSO4, 10 KCl, and 5 glucose; pH 7.4) for the rest. Drugs were applied simultaneously to apical and basal chambers.

Measurements of iron transport. In most cases, uptake, cellular retention, and overall transport of 59Fe-labeled Fe was monitored as previously described (50), with minor modifications. Monolayers were made iron deficient by treatment with DFO (1 mM) for 18 h. Transport was monitored for 15 min to 1.5 h after applying 1 µM 59FeCl3 reduced with 1 mM ascorbate in HEPES buffer to the apical chamber. HEPES buffer with 39 µM human apoTf was the basal solution. The pHs of the apical and basal solutions were 5.5 and 7.4, respectively. Radioactivity in fluids and cells (washed 3x with HEPES buffer containing 1 mM ascorbate) was determined by gamma counting (COBRA II Auto-Gamma; Packard Instruments, Downers Grove, IL). Uptake was calculated as the sum of the radioactivity in the basal fluid and cells; "overall transport" was radioactivity transferred to the basal fluid; "cellular retention" was that in the washed monolayers.

The integrity of the monolayer was checked with phenol red included in 100 µl apical fluid (final concentration of 16 µg/ml) during 59Fe transport. At the end, the absorbance of the basal fluid was measured at 558 nm, using normal basal medium as a blank. No increase in absorbance was detected in the basal fluid at the end of our experiments. Because the absorbance of 16 µg/ml phenol red was 0.700, we should have been able to detect at least 2–3% of the dye crossing the monolayer. We also measured [14C]mannitol leakage and found that it was 1–3% over 6 h and not affected by treating the cells with inhibitors, such as AG10. Because the overall effects we report are well beyond 1–3%, leakage cannot have been a significant factor.

For measurement of apoTf uptake, human apoTf was labeled with 125I using the gentle Iodogen method of Pierce Biotechnology (Rockford, IL). 125I-labeled apoTf was mixed with nonlabeled apoTf to the usual 39 µM final concentration and placed in the basal fluid.

In some cases, studies were carried out at 4°C or inhibitors of different aspects of vesicular transport were added to both apical and basal fluids. Concentrations of these inhibitors were BFA (20–50 µg/ml), nocodazole (5–200 µM), FSBA (0.1–1 mM), and AG10 (500 µM). The 4°C studies were carried out on ice in a CO2 incubator set at 28°C. Plates were continuously kept on an ice slab, and ice-cold buffer was used for the washes.

Release of 59Fe from Caco-2 cell monolayers across the apical and basolateral membranes was followed using DFO-treated monolayers preloaded from the apical side with 59Fe-ascorbate (1 µM-1 mM) in HEPES buffer for 1–1.5 h. (Approximately 55% of the radioactive iron was taken up and retained by the cells.) After twice being washed with cold 1 mM ascorbate in HEPES and once with HEPES alone to remove surface 59Fe, cells were incubated for 15 min-1.5 h in fresh HEPES buffer (apical) and HEPES with 39 µM apoTf (basal), and the appearance of radioactivity in the apical and basal fluids was monitored. Release was calculated relative to the 59Fe present in cells at the start of the release period. (This intracellular 59Fe was determined by difference, based on 59Fe recovered in the washes after the preloading.)

Values for iron transport and distribution were corrected for cell number by measuring the protein content of the cell layers by the Bradford assay, with bovine serum albumin as the standard. Thus transport values are expressed primarily as picomoles per milligram of cell protein. Results are expressed as means ± SD for the number of determinations indicated. Statistical analysis of the data was by one-way ANOVA. P values of <0.05 were considered significant.

Measurements of Cu and glucose release from preloaded monolayers. This was done in the same manner as for Fe release, except that cells were preloaded with 1 µM 64Cu-labeled Cu(II) dihistidine or 5–100 mM [14C]glucose, and release into the basal and apical media was calculated based on radioactivity measured in a gamma counter (64Cu) or by scintillation counting (14C).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of apoTf on iron transport: apoTf enhances iron transport and its release across the basolateral membrane. Intestinal iron absorption occurs in three steps. Iron crosses the apical (brush border) membrane, transfers across the intracellular space, and then crosses the basolateral membrane. Ma et al. (29) showed that apoTf added to the basal chamber of Caco-2 cell monolayers in Transwells traveled to perinuclear endosomes and that DMT1 from the apical membrane went to the same compartment. This colocalization was found to be specific for apo (as opposed to holo-)- Tf. After colocalization, apoTf and DMT1 returned to the basolateral and apical membranes, respectively. Thus it was postulated that iron (internalized via DMT1) was transferred to apoTf (entering from the basal fluid) in perinuclear endosomes and carried to the basal fluid by apoTf. On the basis of their findings, our first task was to confirm the involvement of apoTf in Caco-2 cell iron absorption and to pinpoint at what steps it might be involved.

Standard transport of 59Fe (II) through the monolayers was monitored for 90 min in the presence and absence of apoTf in the basal fluid. As previously reported by our laboratory (50) and others (2, 4), apoTf stimulated overall transport of Fe across the monolayer (Fig. 1). We also found it significantly enhanced brush-border uptake (Fig. 1). Enhancement averaged 20 and 50% for uptake and overall transport, respectively (P < 0.001). As a consequence, there was a threefold reduction in cellular retention with apoTf. This implies that apoTf is part of the iron transport system of this intestinal cell model and that its main effect is to enhance release of iron from intestinal cells to the blood.



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  		Fig. 1. Stimulation of iron transport in Caco-2 cell monolayers by apotransferrin (apoTf). Caco-2 cell monolayers with tight junctions were made iron deficient by overnight treatment with desferrioxamine (DFO). Uptake, cellular retention, and overall transport of 1 µM 59Fe (II) applied to the apical chamber were followed for 1.5 h, with (black) and without (white) apoTf in the basal chamber. Data (in pmol/mg cell protein) for 2 experiments are given as means ± SD (n = 4). (Each Transwell contained ~0.67 mg cell protein.) Average uptakes, cellular retention, and overall transport of 59Fe by control monolayers were 30, 6, and 26% of dose, respectively. *P < 0.001 for difference from without apoTf.

 
To confirm this concept, release of 59Fe from preloaded cells, in the absence and presence of apoTf in the basal fluid, was also monitored. Iron-deficient cells were preloaded apically with 59Fe for 1.5 h. [Half (47%) of the 59Fe was taken up.] Consistent with the results in Fig. 1, basal efflux of cellular radioactive iron over 60 min was significantly enhanced by apoTf (Fig. 2A), going from 6.5 to 9% of that initially in cells. The effect (over 60 min) was not as great as that over 90 min (Fig. 1), presumably because of the relatively slow cycling of apoTf into and out of the Caco-2 cells (2, 33). This was verified by following 59Fe transport for several hours (Fig. 2B), during which more than a doubling of the rate of release was observed. These substantial effects of apoTf on iron transport across the basolateral membrane imply that endo-exocytic cycling of apoTf is involved. Uptake was also affected, suggesting that apoTf endocytosis (from the blood side) also signals changes at the brush border.



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  		Fig. 2. Enhancement of basolateral iron release by apoTf. A: after uptake of 1 µM 59Fe (II) by iron-deficient Caco-2 cells for 1.5 h, during which 47% was incorporated, release of the 59Fe into the basal chamber was monitored for 1 h in the absence (white) and presence (black) of apoTf in the basal fluid. The values are means ± SD (n = 4) expressed as pmol 59Fe/mg cell protein. *Mean differences were significant (P < 0.005). B: overall transport (from apical to basal chamber) by Caco-2 cell monolayers (as in Fig. 1) monitored over several hours. Data show 59Fe appearing in the basal medium [means ± SD (n = 6–9) for 2 experiments] in the presence (top line) and absence (bottom line) of 39 µM apoTf added to the basal chamber. *Differences were statistically significant (P < 0.001).

 
To further examine the relationship between apoTf and basolateral Fe transport, we measured rates of uptake of 125I-labeled apoTf (39 µM) from the basal medium. Uptake rates (initial rates) were 1.2 pmol/min. This compared with rates of ~0.1 pmol/min for basolateral Fe release, indicating that more than enough apoTf was being internalized to account for the increased Fe released. [Indeed, the results indicated that only a small portion of the apoTf that entered was connecting with iron coming across the brush border.]

Effects of lowering temperature on uptake and release of iron from Caco-2 cell monolayers: iron uptake across the apical membrane is energy dependent and probably vesicular. To obtain additional evidence that iron uptake at the brush border (enhanced by apoTf) was at least partly vesicular, the effect of cooling to 4°C on apical uptake of 59Fe(II) was examined. Microtubules are essential for endo- and exocytosis of vesicles, and it had been demonstrated that they are destroyed at 4°C (21). As shown in Fig. 3, initial rates of uptake were markedly lower at 4 vs. 37°C. At 37°C, cells took up 50% of the total 59Fe applied within the first 15 min, compared with about 5% at 4°C. After the initially rapid 59Fe uptake at 37°C, apparent uptake slowed and accumulation plateaued, probably reflecting an increased exit of 59Fe across the basolateral membrane and reduced levels of 59Fe for uptake in the apical fluid. After 15 min at 4°C, accumulation of 59Fe also slowed down and even at 90 min was <10% of dose (compared with 70% at 37°C). This dramatic reduction in 59Fe uptake is consistent with the destruction of microtubules and reduction in ATP needed for vesicular transport.



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  		Fig. 3. Reduction of iron absorption at low temperature. 59Fe(II) uptake (accumulation) by iron-deficient Caco-2 cell monolayers was measured at 4 ({bullet}) and 37°C (X) over 90 min in the presence of apoTf in the basal medium. Values are means (pmol/mg cell protein) ± SD (n = 3–5). Average initial rates at 4 and 37°C were 0.4 and 2.6% of dose/min, respectively. *Comparative values at all time points were significantly different (P < 0.001).

 
Basal iron efflux requires vesiculation and energy. To demonstrate that basal efflux of iron also depended on energy and vesicular transport, the same iron-release studies described in Fig. 2A were performed and compared at 4 and 37°C, with measurements from 15 to 60 min in the presence and absence of apoTf. Release of Fe to the basal fluid in the presence of apoTf was markedly reduced by cooling: at 37°C, it reached 8% of total cellular 59Fe (or ~6 pmol/mg cell protein) after 60 min, compared with <1% at 4°C.

To determine whether temperature-dependent efflux of iron was linked to apoTf alone, parallel studies were done without apoTf in the basal medium. At 4°C, basal iron release was still markedly reduced by cooling, falling from ~6% of cellular 59Fe to ~1% at 60 min. Thus again, a portion of basolateral iron release depended on apoTf. However, our results implied that the non-apoTf-dependent process by which Fe traverses the basolateral membrane also requires energy and/or is vesicular.

Some iron exits across the apical membrane via an energy-independent mechanism that does not involve apoTf. In the course of the studies just described, efflux of Fe across the apical membrane was also followed. Surprisingly (Fig. 4, A and B), the Caco-2 cells released more iron into the apical than basal fluids: ~20 vs. 4–6 pmol/mg cell protein, respectively. (As usual, there was greater efflux in the presence of apoTf.) Time-course studies showed that apical efflux rates (Fig. 4, C and D) were considerably higher than basolateral rates, whether or not apoTf was present in the basal fluid. Over 60 min, 59Fe efflux was a bit higher in the presence of apoTf (Fig. 4, C vs. D).



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  		Fig. 4. Role of apoTf in basolateral and apical efflux of iron. A and B: cells were treated as in Fig. 3, and 60-min release of cellular 59Fe into the basal (A) and apical (B) chambers was compared at 37°C, with (open bars) and without (filled bars) apoTf in the basal fluid. C and D: apical efflux was also measured at 4 ({circ}) and 37°C ({bullet}) in the presence (C) and absence (D) of apoTf. Values are means ± SD (n = 4 for A and B; n = 7–8 for C and D) and expressed as in Fig. 3. *P < 0.005, **P < 0.01 for difference from no added apoTf.

 
Of additional interest was the observation that bringing the temperature to 4°C had little or no effect on apical Fe efflux rates (Fig. 4, C and D). Over 15 min, no significant reduction in rate was seen at 4°C. Initial rates were the same at 37 and 4°C (1%/min) and were unaffected by apoTf. However, at 4°C and later times, the accumulation of 59Fe in apical fluid was not maintained and began to decline if apoTf was present, suggesting that the cycling in of apoTf permitted reuptake of the iron. Without apoTf, the pattern of apical 59Fe release was identical at 4 and 37°C. Low temperature and apoTf thus had no statistically significant effects on apical efflux of iron from the Caco-2 cell monolayers.

To further explore the mechanism underlying the apical release of iron, we first examined the effects of adding 1 µM nonradioactive Fe(II)-ascorbate to the apical medium (the concentration used during our normal uptake studies), in line with the concept that this would lower the chemical Fe gradient (from inside to outside the cell) that might be driving Fe efflux. As shown in Fig. 5A, there was a small but significant inhibition. However, a 10-fold higher concentration of Fe(II) in the apical fluid did not increase the efflux inhibition (Fig. 5B), implying more complexity (such as that the form of Fe being released was not identical to that in the apical medium).



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  		Fig. 5. Inhibition of the apical release of cellular 59Fe by nonradioactive Fe in the apical medium. After uptake of 1 µM 59Fe (II) by iron-deficient Caco-2 cells for 2 h, release of the 59Fe into the apical chamber was monitored for 15 min. A: release in the presence (filled bar) of 1 µM nonradioactive Fe(II) ascorbate in the apical medium or in HEPES buffer alone (open bar) or with ascorbate (shaded bar). B: release in the presence of 10 µM nonradioactive Fe(II) ascorbate (black bar) in the apical medium or with HEPES buffer + ascorbate (open bar). Release values are means (±) SD (n = 6–9) expressed as pmol 59Fe/mg cell protein. *Mean differences were significant (P < 0.03).

 
We also examined the apical efflux of glucose at 4°C, which involves facilitated transport down a chemical gradient. For this, cells were preloaded with [14C]labeled for 30 min (at 37°C), washed thoroughly with cold HEPES medium, and allowed to release 14C at 4°C. Substantial amounts of the retained 14C were released, and the release was concentration dependent. Thus with preexposure of cells to 10 mM glucose (during which 4.8% was taken up), 4% of the 14C corresponding to 13.4 nmol glucose (or metabolites) was released back into the apical fluid in 15 min, at 4°C. With 100 mM glucose preexposure (where uptake was 2.2%), 7% of the internal 14C (corresponding to 108 nmol glucose) flowed back out in 15 min, even at 4°C. In the case of the 59Fe release, the absolute amounts were of course much less.

Finally, we examined whether lowering the temperature would affect release of another metal ion (Cu) from the Caco-2 cell monolayers. In the case of Cu, basolateral Cu release is thought to depend on vesicular transport and exocytosis involving the MNK protein (37). This was markedly inhibited at 4°C, going from 24,400 ± 9,400 (mean ± SD; n = 9) to 5,000 ± 5,500 cpm/mg cell protein (n = 11), a drop of 80%. In contrast, and similar to our findings for Fe, apical release was robust (at 29,900 ± 7,900 cpm/mg cell protein; n = 7) and was not significantly affected by the drop in temperature (22,200 ± 8,500; n = 9). Our findings suggest that apical iron (and copper) release occurs via diffusion down a concentration gradient facilitated by an unknown carrier and does not depend on energy and apoTf.

Effect on iron absorption of reagents interfering with vesicular transport. In addition to vesicular transport, many other cellular processes (including ATP synthesis) are slowed at low temperatures. To specifically interrupt vesicular transport, we thus used reagents that depolymerize microtubules, inhibit vesicle formation from the plasma membrane, alter protein sorting, and/or prevent membrane fusion between vesicles and endosomes. In each case, Caco-2 cell monolayers were pretreated with a drug, and standard 59Fe transport studies were carried out in its continued presence.

Apical uptake and basal release of 59Fe are reduced 50% by nocodazole and fluorosulfonylbenzoyladenosine. Nocodazole is an antimitotic agent that depolymerizes microtubules. It also causes dispersal and tubulation of the Golgi apparatus (44), because this apparatus depends on microtubules for alignment and positioning (43). Fluorosulfonylbenzoyladenosine (FSBA) is an ATP analog that can interfere with sequestration of receptors into clathrin-coated pits at the beginning of endocytosis. Receptor sequestration requires phosphorylation of Thr156 in the µ2-subunit of an adaptor complex (AP2) (36). FSBA binds to this Thr and prevents clathrin-dependent internalization of the Tf receptor (TfR) complex with Tf in HeLa cells (56). We thus expected with nocodazole to inhibit transport of vesicles along microtubules (through the intracellular space) and with FSBA to prevent endocytosis at a much earlier stage (possibly at formation of a vesicle at the plasma membrane).

Caco-2 cell monolayers were preincubated for 1.5 h in DMEM supplemented with FBS (1%) and nocodazole (5–200 µM) or with 100 µM-1 mM FSBA for 2 h (doses used by others for cell culture studies). Standard 59Fe transport studies were initiated by addition of 59Fe to the apical pretreatment buffer; and transport was followed for 1 h with apoTf in the basal fluid. Uptake and overall transport (basolateral release) of 59Fe were each decreased ~50% by the various concentrations of nocodazole (Fig. 6A) and FSBA (Fig. 6B). Maximum effects were already observed at the lowest doses, so data for all doses were combined. These results strongly support the notion that iron absorption involves endocytic steps at both the apical and/or basolateral membranes, and they suggest that ~50% of intestinal iron transport may be vesicular. In both cases, the decreases in uptake and overall transport mostly balanced each other out, so that there was only a small or no change in cellular retention of the 59Fe.



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  		Fig. 6. Effect of nocodazole and 5'-(4-fluorosulfonylbenzoyl)adenosine hydrochloride (FSBA) on uptake and overall transport of iron. Caco-2 cell monolayers were preincubated either with nocodazole (A; 5–200 µM in 1% FBS DMEM) for 1.5 h or with FSBA (B; 0.1–1 mM in HEPES buffer) for 1.5–2 h. After preincubation, 59Fe (1 µM) and apoTf were added to the existing apical and basal fluids, respectively, for standard 1-h 59Fe transport studies. Values are for 2 separate experiments and given as pmol/mg cell protein (means ± SD; n = 8–13 for A and n = 3–4 for B). Average uptakes, cellular retention, and overall transport of control monolayers were 21, 4, and 14% of dose, respectively, for the nocodazole studies. For the FSBA studies, they were 24.8, 0.2, and 24.5% dose, respectively. Because maximum inhibition was achieved at the lowest dose, data from all doses were combined. *P < 0.005, **P < 0.02, ***P < 0.05 for differences from control (untreated) cells.

 
Brefeldin A slightly reduces apical uptake and basolateral transfer of 59Fe. In Caco-2 cells, apoTf travels from the basal to the apical domain, whereas holoTf (diferric Tf) cycles within the basal domain (29, 32). However, it is uncertain which endosomes are involved in the apoTf-recycling path. We investigated the effects of brefeldin A (BFA) because this fungal metabolite was shown to alter sorting of TfR in Caco-2 cells (46), interfere with dynamin-dependent TfR recycling in HeLa cells (45), and disrupt the Golgi apparatus (27). In the Caco-2 cells, BFA had also been reported to cause 5–15% of TfR to route to the Golgi via an unknown microtubule-independent mechanism distinct from the recycling paths (half-time of 1.5 h) (21). In our studies, Caco-2 cell monolayers were pretreated with BFA (20–50 µg/ml) for 1.5 h as well as during standard 2 h 59Fe transport studies, with apoTf in the basal fluid. As shown in Fig. 7, BFA had small but statistically significant negative effects on uptake, cellular retention, and overall transport of Fe. The decrease in uptake was only 10% (P < 0.001); that in overall transport was 6% (P < 0.01). These reductions were accompanied by a 10% decrease in cellular 59Fe retention (P < 0.005). This implies that at least a small portion of uptake and overall Fe transport may involve endosomes within either the dynamin-dependent TfR recycling path or the pathway leading to the Golgi.



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  		Fig. 7. Slight inhibition of iron transport by brefeldin A (BFA). Caco-2 monolayers were pretreated for 1.5 h, with BFA (20–50 µg/ml in HEPES buffer), followed by standard, 2-h 59Fe transport studies, with 59Fe and apoTf applied directly to the existing apical and basal medium, respectively. The data are expressed as in Fig. 6 (n = 22–29). *P < 0.001, **P < 0.005, ***P < 0.01.

 
Tyrphostin A8 (AG10) inhibits uptake independent of apoTf and enhances the stimulatory effect of apoTf on iron absorption. Unlike other tyrphostins, tyrphostin A8 acts as an inhibitor of a GTPase instead of a tyrosine kinase. Like BFA, it missorts TfR during its vesicular transport from the basolateral to the apical membrane in Caco-2 cells (46). Vesicular transport involves membrane fusion of vesicles and endosomes, and this process requires small GTPases (Rab proteins). Rab17 is restricted to epithelial cells (28) and found in the apical endosomes of polarized cells [such as MDCK (20) and Eph4 (49)]. Apically and basolaterally internalized proteins, including TfR, reach these Rab17-enriched apical endosomes (20, 49). Such apical endosomes, accessible to both apically and basolaterally internalized molecules, were identified in Caco-2 cells (19, 23) as well as MDCK cells (8). Apically endocytosed DMT1 and basolaterally internalized apoTf colocalized in the apical/perinuclear endosomes (29). Therefore, AG10 seemed a good choice to interrupt vesicular transport mediated by DMT1 and apoTf.

As with previous studies, AG10 was applied to both sides of the Caco-2 cell monolayers at the same time as the 59Fe was applied apically to measure transport. Transport was followed over 6 h in the absence and presence of apoTf in the basal medium. (Analysis with phenol red confirmed the integrity of the monolayers at the end of the studies.) In the absence of apoTf (Fig. 8A), AG10 decreased uptake and cellular retention of 59Fe 35 and 45%, respectively (P < 0.001). Reduction of the former explained the latter, because there was no statistically significant change in overall transport. Hence, AG10 inhibited uptake of 59Fe, but not its transfer across the basolateral membrane.



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  		Fig. 8. Effect of AG10/tyrphostin A8 on iron absorption. The standard 59Fe transport was carried out over 6 h in DMEM in the absence (A) or presence (B) of apoTf in the basal medium. AG10 (500 µM in DMEM) was applied to the monolayers at the same time as the 59Fe used to monitor transport. The data are presented as in Fig. 6 (n = 7–8 for A; n = 9–10 for B). Without apoTf, average uptakes, cellular retention, and overall transport for control monolayers were 52, 45, and 7% of dose per 6 h, respectively; with apoTf, they were 64, 47, and 16% of dose, respectively. *P < 0.001 for difference from untreated (control) cells.

 
To see the effect of AG10 on vesicular transport mediated by apoTf, apoTf was added to the basal medium. As shown in Fig. 8B, AG10 strikingly increased apoTf-mediated overall transport of 59Fe, which doubled. Uptake remained inhibited (25%; P < 0.001). Cellular retention was thus even lower than in the absence of apoTf (P < 0.001). These considerable effects of AG10 indicated not only that vesicular processes were involved in iron transport, but lent further credence to the concept that apoTf plays a role in basolateral efflux of iron from the enterocyte.

To better understand the effects of apoTf and AG10, values for cellular retention and overall transport of 59Fe were recalculated in terms of the amounts of radioactive iron that had been absorbed (Table 1). Without AG10, apoTf shifted 13% of the absorbed 59Fe from cellular retention to overall (basolateral) transport; with AG10, it shifted ~37%. This confirmed that despite inhibiting uptake, AG10 enhanced the stimulatory effect of apoTf on intestinal iron absorption.


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  		Table 1. AG10 enhances the stimulatory effect of apoTf on iron transport. Data from studies shown in Fig. 8, A and B were recalculated as mean percentages of the 59Fe in cells at the start of the release process

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
With the use of polarized Caco-2 cell monolayers with tight junctions, grown on filters, we have shown in several different ways that apoTf influences transport of iron across the enterocyte. Because these monolayers are by now an accepted model for studies of intestinal mucosal function, our findings imply that apoTf also influences and participates in dietary iron absorption. In the studies reported here, we consistently found significant stimulatory effects of apoTf, particularly on efflux of apically absorbed iron from the monolayers across the basolateral membrane (Figs. 1 and 5C). The effects were more readily apparent during transport studies over longer time periods. Thus basolateral transport was stimulated 20–30% over 60 min (Figs. 2A and 4A), ~50% over 90 min (Fig. 1), and >100% over several hours (Fig. 2B). This agrees with previous reports from the Glass-Nunez groups (2, 4, 34), who also saw stimulations of up to 100%. It is also consistent with reports that the half-time for cycling of apoTf into and out of the Caco-2 cells from the basal medium is ~60 min (4, 30, 33). These observations are also consistent with the proposed model that apoTf enters from the blood plasma at the basal surface of the enterocyte (which could only happen through endocytosis); it translocates to the upper part of the enterocyte in vesicles that fuse with DMT1-containing endosomes and then trafficks back to the basal membrane with the purpose of producing ferric Tf for release into the blood.

Our data also showed a small but significant enhancement by apoTf of iron uptake. This stimulation by basolaterally added apoTf at the brush border is harder to explain but might be the result of coordination between the vesicular processes that cause DMT1 and apoTf to enter endosomes at opposite sides of the monolayer. We found that 90-min exposure to apoTf in the basal medium resulted in 20% more iron uptake (Fig. 1). Stimulation of uptake by apoTf was not observed by Glass and colleagues over 60 min (2), but because of experimental variability, a small effect might not have been detected.

In all our studies in which apoTf was implicated in promoting Fe transport, lowering the temperature to 4°C had profound inhibitory effects. Basolateral iron efflux was reduced 7- to 12-fold. This is consistent with what we know about the biochemistry of endocytic cycling and vesicular transport, which require considerable energy and the use of microtubules that permit vesicles to travel along the cytoskeleton. A reduction in temperature slows metabolic/enzymic reactions, lowering rates of energy metabolism and ATP (and GTP) production. Microtubules actually disassemble at 4°C (21), making endocytic trafficking impossible. Enzyme activities (and perhaps also transporters that have metal ion channels) tend to reduce their activities by one-half for every 10°C. Thus we would have expected the 30°C drop in temperature to result in an 88% (or 8.3-fold) drop in transporter activity and an even greater drop if there was a requirement for ATP, as in the case of vesicular trafficking. The generally greater than 10-fold drop observed thus also supports involvement of such processes.

The impact of adding apoTf to the "blood" side of Caco-2 cell monolayers was also evident in experiments in which we tested the effects of drugs that interfere with normal vesicular processes. This was particularly clear in our studies with AG10, which is thought to inhibit the normal sorting of apical endosomes (46), perhaps by binding to Rab17 and preventing GTP hydrolysis. AG10 has been reported to shunt Tf receptors to the apical membrane (76); but because apoTf is not likely to enter through this receptor, we would have expected a different response. In our case, it markedly enhanced apoTf-dependent basolateral Fe efflux. This not only suggests that AG10 was causing a missorting of endosomes (perhaps a blockage to trafficking of apoTf endosomes to the apical regions of the cell), but also shows that apoTf endosomes entering from the basolateral membrane are still capable of moving Fe from the cell into the blood through vesicular trafficking. In addition, it highlights the importance of apoTf for the transfer of iron across the basolateral membrane.

In all, the data from our studies indicate that apoTf clearly does become involved in the shuttling of iron into, across, and out of the intestinal epithelial cell and is particularly helpful in releasing it to the blood. As already indicated, our results agree with and expand on the original observations of Nuñez and Glass and their collaborators (2–4, 29, 30, 33, 34). They also agree with our own previous report showing stimulation of uptake as well as overall transport of iron in Caco-2 cell monolayers (50). Although supportive of the involvement of apoTf, our results also indicate that processes dependent on this plasma protein are not the only ones capable of transferring iron across either end of the enterocyte; i.e., there are other ways for iron to cross the brush border and basolateral membrane that do not involve apoTf. More surprisingly perhaps, some of our data suggest that apoTf-independent basolateral Fe transfer also at least partly involves vesicular transport, because it was greatly impacted by cooling. This would be consistent also with increasingly common reports that transporters and other factors vital for intestinal iron absorption are either primarily or partly detected in endosomes (5, 9, 22, 29, 30, 42).

In contrast to basolateral release, our data imply that apical release of iron is not vesicular. Cooling to 4°C had virtually no effect, and as with glucose, substantial amounts of newly absorbed 59Fe went back out across the brush border at this temperature. This suggests that Fe diffuses across the brush border down a concentration gradient. [One is tempted to speculate that ferroportin, lately found also to be associated with the brush border (30, 42), might be involved.] However, addition of external Fe(II) (in the form of the ascorbate complex) had only a small (but significant) inhibitory effect; but that is not surprising, because the form of Fe released (and descending the gradient) is likely to be different. Interestingly, release of internalized copper across the apical brush border also was not affected by lowering the temperature, although the process of basolateral transfer, which is known to depend on vesicular processes, was affected as severely as was that for iron, underscoring the role of vesicular transport in iron absorption.

The stimulatory effects of apoTf in themselves implicate endo- and exocytosis in intestinal iron absorption. Our findings of profound inhibition by low temperature and marked changes induced by specific inhibitors of vesicular processes lend additional strong support to this notion. All of the inhibitors tested significantly altered either uptake and/or basolateral iron transport in the monolayers. The largest effects were obtained with nocodazole and FSBA, which inhibited both uptake and basolateral transfer by ~50%. Thus half of the iron absorption might be vesicular. Part of this appears to involve the cycling of apoTf into and out of the enterocyte and is consistent with the concept that it migrates to apical regions of the cell (in vesicles, along the cytoskeleton), fuses with DMT1-containing endosomes from the brush border to take on iron, and cycles back to the blood side as ferric Tf. But again, even in the absence of apoTf, some kind of vesicular cycling was occurring and responsible for overall transport across the monolayer to the "blood." The data we obtained with specific inhibitors indicate that endocytosis is also important at the brush border. Nocodazole, FSBA, and AG10 each inhibited iron uptake by about half. It is unclear whether this is related to trafficking of DMT1. One has assumed that DMT1 operates as a kind of channel in the brush-border membrane, but its retreat into endosomes (22, 29, 30) and their coalescence with apoTf-containing vesicles may mean that, here, it simply acts as an iron binding protein to mediate vesicular Fe transport across the cell and into the basal medium. Nocodazole and FSBA both also inhibited basolateral iron transport by ~50%. Because ~50% of this process may depend on apoTf cycling, it seems likely that this was the main target of these drugs and explains their effects.

In combination with the observations of others, our results indicate that vesicular transport is responsible for a major part (at least half) of intestinal iron absorption, as modeled by the Caco-2 cell monolayers, and that endocytosis of iron at the brush border is occurring as well as exocytosis across the basolateral membrane, some, but not all, of which involves apoTf if available. This still leaves room for nonvesicular transport of iron across both cell borders, most likely involving brush-border DMT1 (but possibly also other transporters) at the apical end, and ferroportin (IREG/MTP1) at the basal membrane.


   ACKNOWLEDGMENTS
 
This work was supported in part by U.S. Public Health Service Grant No. RO1 DK-53080.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Maria C. Linder, Dept. of Chemistry and Biochemistry, California State Univ., Fullerton, CA 91834–6866 (e-mail: mlinder{at}fullerton.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.


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