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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.33 n.2 Santiago  2000

http://dx.doi.org/10.4067/S0716-97602000000200013 

The cellular mechanisms of body iron homeostasis*

MARCO T NUÑEZ, MARCO A. GARATE, MIGUEL ARREDONDO, VICTORIA TAPlA
AND PATRICIA MUÑOZ

Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. Instituto Milenio de Estudios Avanzados en Biología Celular y Biotecnología, Santiago, Chile

ABSTRACT

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transterrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.

KEY TERMS: metal ions; intestinal absorption; ferritin; transferrin; homeostasis

INTRODUCTION

Iron (Fe) is a nutrient essential for life. It supports fundamental biological functions involved in the handling and transport of oxygen, nitrogen fixation, detoxification, photosynthesis, and synthesis of DNA (Crichton and Ward, 1992). Iron is potentially toxic to cells (Wrigglesworth and Baum, 1980). Its toxicity derives from the catalytic production of free radicals through the Fenton and Haber-Weiss reactions (Wardman and Candeais, 1996). The essential/toxic nature of iron has resulted, through evolution, in molecular mechanisms that secure cell and body iron homeostasis.

Cellular iron homeostasis is achieved through the activities of IRP1 and IRP2, cytosolic proteins that bind to structural elements named IREs, which are present in the untranslated region of mRNAs that codify for proteins involved in iron metabolism, such as ferritin and the transferrin receptor (Leibold and Munro, 1988; Mullner et al., 1989; Klausner et al., 1993; Samaniego et al., 1994; Iwai et al., 1998). The activities of IRP 1 and IRP2 respond to cellular Fe, but through different mechanisms. Low levels of intracellular iron activate IRP1 to bind to and stabilize the transferrin receptor (TfR) mRNA and to bind to ferritin mRNA, thus diminishing its translation. High levels of intracellular iron inhibit the IRE-binding activity of IRP1. IRP2 is always active to bind to IRE, but its mass is regulated through Fe-induced IRP2 ubiquitination and proteasome degradation (Iwai et al., 1998). Overexpression of a mutant IRP1 constitutively active in binding to IRE produces cells that express high levels of TfR despite of iron repletion (Derusso et al., 1995). This was the first direct demonstration of IRP1 involvement in the expression of proteins of iron metabolism.

Body iron levels are also regulated. In healthy individuals, iron losses are closely compensated by increased intestinal absorption (Flanagan 1989; Beard et al., 1996). Current models of iron absorption by intestinal epithelia postulate that incoming iron is incorporated in a pool of chelatable iron (Flanagan 1989; Beard et al., 1996; Conrad and Umbreit 1993; Wood and Han 1998). Iron from this pool would either bind to ferritin, mobilferrin and unknown iron-binding proteins, or it would be transferred to blood plasma proteins through the basolateral membrane of the enterocyte (Flanagan 1989; Beard et al., 1996; Conrad and Umbreit 1993; Wood and Han 1998). Ferritin, in particular, has been proposed to regulate iron absorption. Cellular iron retention was found to be inversely proportional to the level of cell ferritin (Mattia et al., 1986). Intestinal epithelial cells respond to a fall in body iron stores by increasing the absorption of dietary Fe, so the extent of iron transport through the intestinal epithelium is inversely related to the content of body iron stores. Caco-2 cells have been described as an excellent in vitro model of human enterocytes (Alvarez-Hernandez et al., 1994; Hillgreen et al., l995). Grown in bicameral inserts, they show apical iron uptake and transepithelial iron transport (Alvarez-Hernandez et al ., 1991; Núñez et al., 1994; Tapia et al., 1996), and transferrin-mediated basolateral iron uptake (Núñez et al., 1996). Caco-2 cells reduce Fe3+ to Fe2+ in the apical medium, and this reduction correlates with increased iron uptake (Núñez et al., 1994; Han et al., 1995). Moreover, the levels of intracellular iron control the mechanisms responsible for the regulation of iron absorption through as yet unknown mechanisms (Tapia et al., 1996). Intestinal epithelia cells have an active IRE/IRP system. IRP activity was found normal in individuals with hemochromatosis (Flanagan et al., 1995). On the contrary, individuals with idiopathic hemochromatosis or iron deficiency anemia have decreased duodenal ferritin expression (Pietrangelo et al., l 992). Hence, the role of IRE/IRP system in the regulation of intestinal iron absorption remains undetermined.

We found that Caco-2 cells cultured to different intracellular iron concentrations work in a concerted manner to regulate IRP I and IRP2 activities, apical iron uptake activity, ferritin levels and transferrin receptor density. Interestingly, a fraction of the IRP-2 activity in Caco-2 cells was unresponsive to iron overload, thereby producing basal levels of apical iron uptake and TfR. Overexpression of the IRE resulted in cells with high levels of ferritin, low levels of the labile (reactive) iron pool, and a high and deregulated transepithelial iron transport. These results demonstrate that intestinal iron absorption is regulated by the IRE/IRP system.

METHODS

Reagents. The polyclonal antibody against ferritin used was from DAKO (Carpinteria, CA). Phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, pepstatin, A, 3-[N-morpholino] propanesulfonate (MOPS), diethylemetriaminepentaacetate (DTPA), and salts were from Sigma Chem. Co., St. Louis, MO. Fetal bovine serum, Dulbecco’s-modified Eagle medium, and low-iron Iscove medium were from ClIBCO Laboratories (Grand Island, NY). 59Fe and 55Fe in the ferric chloride form were from New England Nuclear (Boston, MA). Culture plasticware and Transwell bicameral inserts were from Costar (Cambridge, M A) . To eliminate contaminant Fe, all buffer solutions were filtered through Chelex- 100 (Sigma). Isonicotinoyl hydrazone (SIH) was the kind gift of Dr. Prem Ponka, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Canada.

Cell culture and 55Fe loading. Caco-2 cells (ATCC # HTB37, Rockville, MD), were cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum. The culture medium was changed every 2-3 days. Cells; grown for 13-15 days were used. Cells with different levels of intracellular iron were obtained as described by Tapia et al., 1996.

Briefly, cells were plated at 5 x 105 cells/ flask (COSTAR, Cambridge, MA) and incubated for a week in Iscove low iron medium (GIBCO Laboratories, Grand Island, NY) and 10% low iron serum ([Fe] < 0.2 µM) (Alvarez-Hernandez et al., 1991), supplemented with variable (1 -10 mM) concentrations of Fe3+ as the complex 55FeCl3-sodium nitrilotriacetate (55FeNTA1, 1:2 molar ratio). During this period the cells reached confluence, with 4-6 x 106 cells/25 cm2 flask. The cells were trypsinized and seeded at a density of 5 x 105 cells/flask and cultured as above. After a week of culture the cells had reached equilibrium with 55Fe, so that their intracellular iron concentration could be estimated from their 55Fe radioactivity (Tapia et al., 1996). The values obtained for total intracellular iron were (µM): 14 ± 4, 17 ± 5, 62 ± 8, 88 ± 24, 278 ± 39 and 352 ± 61 for 0.9, 1.4, 2.4, 2.9, 5.4 and 10.4 µM iron added to the culture medium, respectively (mean ± SD of 5 experiments).

59Fe transport studies. Cells obtained from the second trypsinization described above wore plated onto 0.33 cm2 inserts (pore size 0.4 mm, Transwells, COSTAR). The geometry of the inserts defines two culture chambers separated by a porous polycarbonate membrane. Cells seeded from the upper chamber attach and grow on the polycarbonate membrane, forming, after 5-6 days, a polarized monolayer of cells with the apical pole facing the upper chamber and the basal pole connected to the lower chamber through the porous membrane. Insert-growth cells were cultured for 14 days in 55Fe-containing media as before, with a change of media every 2-3 days. Measuring the transepithelial electrical resistance with an EVOM epithelial voltohmmeter (World Precision Instruments, Sarasota, FL) monitored the formation of the cell monolayer in the inserts. For transport studies cells were washed 3 times with PBS and incubated at 37 ºC for varied times ( 15-120 min) with 5 µM 59Fe3+, as the 59Fe-NTA (1:2.2 mol: mol) complex added to the apical medium. The incubation medium was low iron Dulbecco’s-modified Eagle medium. 59Fe uptake was stopped by washing the inserts three times with icecold saline supplemented with 1 mM EDTA. 55Fe and 59Fe radioactivity in the cells and basolateral media was measured in a dualchannel beta counter (Packard Instrument, Meriden, CT).

Cell extracts. To prepare cell extracts, cells were treated with lysis buffer (50 µl per 1 x 106 cells of 10 mM MOPS, pH 7.5, 3 mM MgCl2, 40 mM KCI, I m M PMS F, 1 0 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.7 µg/ml pepstatin A, 5% glycerol, 1 mM dithiothreitol, 0. 1% Triton X-100). The mixture was incubated for 15 min on ice and centrifuged for 10 min at 1,000 x g. The supernatant was stored at -70 ºC.

Western blot analysis of IRPs. Proteins were separated by SDS-polyacrylamide gel electrophoresis using the Laemmli method. Immunodetection was performed using a polyclonal anti IRP antibody (Biosonda, Arredondo et al., 1997) and a chemoluminiscence kit (Amersham ECL Western blotting kit).

Subcloning of IRE and cell transfections. IRE sense and antisense DNA were obtained by synthesis of the nucleotide sequence corresponding to the human H ferritin IRE (Mullner et al., 1989; Klausner et al., 1993), flanked by BamHI and XhoI restriction sites. The main IRE segment was 5' GGATCCTTCCTGCTTCAACAGTGCTTGGACGA-AACCCTCGAG- 3'. After annealing, the probe was cloned into the polylinker of pcDNA3 (Invitrogene, San Diego, CA), previously restricted with BamHI and XhoI (Gibco Life Technologies, Grand Island NY). Sequencing of the construct demonstrated that the IRE was inserted into the plasmid. The plasmid obtained was named pcDNA-IRE. Caco-2 cells grown to half confluence (2-3 days after plating) were transfected with equal amounts of either pcDNA-IRE or pcDNA3 plasmids. Lipofectamine (Gibco) at 5 mL/mg of DNA was used for the transfections. The DNA/lipofectamine mixture was removed after 36 hrs of incubation at 37 ºC. The cells were incubated overnight in DMEM-10% FBS and then changed to a selection medium, DMEM, with 10% FBS plus 0.4 mg/ml geniticin (Gibco G4 18). The cells were grown for 3 passages (one week growth, trypsinization, and re-plating) and then stored in liquid nitrogen.

Northern blot analysis. Total cell RNA was isolated from Caco-2 cells as described (Ausubel et al., 1995), and equal amounts of RNA were electrophoresed in 1.5 % agarose under denaturing conditions. To confirm that each lane contained equal amounts of RNA, the ribosomal content of each lane was determined with ethidium bromide. RNA, transferred to Hybond-N membranes (Amersham), was hybridized with a 32P-labeled IRE probe, consisting of a 28-er antisense sequence of ferritin IRE, end-labeled with [g- 32P]-ATP. As a positive control, the sense ferritin IRE sequence was electrophoresed and hybridized with the above probe.

Measurement of transferrin receptors and ferritin. Transferrin receptor density was determined in cell extracts by an enzymelinked immunosorbent assay (Harlow and Lane, 1988) using OKT9 anti-transferrin receptor monoclonal antibody as primary antibody and peroxidase-labeled goat antimouse IgG (SIGMA Chem. Co., Saint Louis, MO) as a secondary antibody. Intracellular levels of ferritin were determined by a sandwich enzyme-linked immunosorbent assay (Arredondo et al., 1997).

Determination of the labile iron pool. The intracellular pool of reactive iron of Caco-2 cells was determined as described by Epsztejn et al., 1997. Briefly, Caco-2 cells were cultured on cover-slips for 10 days in DMEM, 10% FBS. Calcein-AM (0.5 mM, Molecular Probes, Eugene, OR) was then loaded for 5 min at 37 ºC. After washing the calcein not internalized, the cells were transferred to a cuvette containing 3 ml of MOPS saline (2() mM MOPS-OH, 15() mM NaCl, 1.8 mM CaCl2, 5 mM glucose, pH 7.4) and 5 ml of anticalcein antibody (the kind gift of Dr. Z.I. Cabantchik). After determination of the basal calcein fluorescence (excitation 488 nm, emission 517 nm), the fluorescence of the calcein-iron complex was dequenched by the addition of 100 µM SIH. The increase in fluorescence thus obtained was directly proportional to the iron labile pool. On the occasions indicated in the text, the cells were pre-incubated for 2.5 h, either in Ferich medium (DMEM, 10% FBS + 10 mM Fe-NTA) or in iron-poor medium (Iscove, 10% low-iron FBS) prior to calcein-loading.

Statistical analysis. Variables were tested in triplicate wells, and the experiments were repeated at least twice. Curve fitting was computed using the Prism and InStat programs (GraphPad Software, Inc., San Diego, CA). One-way ANOVA was used to test for differences in means, and post-hoc t test was used for comparison.

RESULTS

IRP activity, apical iron uptake, ferritin, and transferrin receptor levels, respond to similar intracellular iron concentrations. Since both IRP activity and intestinal iron absorption are the function of intracellular iron levels, it was possible that the IRE/ IRP system could be involved in this regulation. We therefore examined the activity of the IRE/IRP system in Caco-2 cells grown to varying levels of intracellular iron (Fig. 1). IRPI activity was dependent on intracellular iron concentrations, reaching nearly null activity at >250 mM total intracellular iron (Fig. 1 A). Interestingly, IRP2 was little responsive to changes in intracellular iron (Fig. 1 A), establishing a basal level of IRP activity that was unresponsive to changes in iron (Fig. 1 B).


Figure 1. IRP activity in Caco-2 cells grown to varied levels of Fe. A: Caco-2 cells were cultured with varied amounts of iron in the culture medium, to which 55Fe was added as a tracer. After three passages, cell extracts were prepared and the IRE binding activity of the extracts was assayed as described in the Methods. B: densitometric analysis of the bands shown in A. C: Integrated (IRPI + IRP2) IRP activity plotted as a function of intracellular Fe.

Apical 59Fe uptake decreased with increasing concentrations of intracellular iron, reaching a basal level that was unresponsive to further changes of intracellular iron (Fig. 2A). Both TfR (Fig. 2B) and ferritin levels (Fig. 2C) changed as a function of intracellular iron in a way that mimicked total IRP activity, indicating that the IRE/IRP system indeed controlled the cellular levels of these proteins in Caco-2 cells. In all cases, the response was similar: a decrease (total IRP activity, 59Fe uptake, TfR levels) or an increase (ferritin levels) in a similar range of intracellular iron concentrations.


Figure 2. Fe transport, TfRs and ferritin levels in Caco-2 cells grown with varied levels of iron in the culture media. Caco-2 cells were grown with varied amounts of iron in the culture medium, to which 55Fe was added as a tracer. After two passages, cells extracts were prepared for the determinaton of TfR (B) and ferritin (C) and levels. Other cells were cultured in bicameral inserts for 14 days, after which apical 59Fe uptake and transhepithelial 59Fe transport were determined( A).

The above data clearly indicated that the apical iron uptake could be regulated by the IRE/IRP system. Reasoning that excess IRE should functionally abrogate the mRNA binding activity of IRPs, we generated Caco-2 cells that over-expressed IRE, and characterized the ability of those cells to regulate iron absorption. Cells transfected with plasmid alone showed one band of approximately 800 nucleotides, most likely corresponding to ferritin mRNA (Boyd et al., 1985), while cells transfected with pcDNA3-lRE evidenced an extra band of about 300 nucleotides (Fig. 3A). This lower band corresponded to IRE, as shown by its hybridization with the anti-sense IRE. Assay of IRP activity showed decreased activity in IRE-transfected cells when compared with cells transfected with pcDNA3 or not transfected (Fig. 3B, compare lines 5 and 6). This decreased activity could be due to the competition of [33P]IRE binding by endogenous IRE, since little or no decrease in IRP activity was observed when 2 mg of cell extract was used per assay instead of the 20 mg used in the present assay (Fig. 3B, lines 1 and 2).


Figure 3. Characterization of IRE-transfected cells., A: Northern blot of pcDNA3-IRE-trasfected cells. Insert-grown Caco-2 cells were transfected with either pcDNA3 or pcDNA3-IRE. After 7 days in culture, total RNA was obtained and separeted in denaturant agarose geks. The proteins were blotted to Hybond-N membranes (Amersham) and were hybridized with a 32P-labeled IRE probe, consisting in the antisense sequence of ferritin IRE, end-labeled with ATP g- 32P. Lane 1, sense IRE sequence; Lane 2. cells transfected with pcDNA3; Lane 3. cells transfected with pcDNA3-IRE.

At low intracellular iron concentrations, cell ferritin in control cells was lower than in IRE-transfected cells, as expected from IRE binding to active IRP and hence inhibiting IRP binding to the IRE motif in ferritin mRNA. Only at high intracellular iron levels was the ferritin content equal in the control and IRE-transfected cells (Fig. 4A). At low intracellular iron levels, Tf receptor levels were about two-fold lower in IRE-overexpressing cells compared with control pcDNA3-transfected cells (Fig. 4B), reflecting the lack of stabilization of TfR mRNA by IRP. Furthermore, TfR levels in IRE-transfected cells did not change in response to changes in intracellular Fe, whereas control cells showed a significant decrease with increasing cell iron (Fig. 4B). These results indicate that in relation to control cells, cells transfected with pcDNA3-IRE up-regulated their ferritin content and down-regulated their TfR levels. Both ferritin and TfR levels remained unchanged, with changes in cellular iron in IRE-transfected cells


Figure 4. Fe, ferritin and TfRs Ievels in IRE-transfected cells. A: total intracellular iron of control and IRE-transfected cells cultured for 7 days in rnediums containing low (0.5 µM) medium (2 µM) or high (5 µM) 55Fe. B: ferritin levels in control and IRE-transfected cells cultured as in A. C: TfR Ievels in control and IRE-transfected cells cultured as in A.

IRE-transfected cells have a deregulated iron transport activity. Considering that IRP-I activity and transepithelial iron transport responded similarly to changes in intracellular iron concentration (Fig. 1), it was of interest to study apical iron uptake and transepithelial iron transport as a function of intracellular iron concentration in IRE transfected cells. For this purpose, control and transfected cells were grown in bicameral inserts, and their capacity for apical 59Fe uptake and transepithelial 59Fe transport activities were compared (Fig. 5). The rate of 59Fe uptake by IRE transfected cells was about twice as large as that of control cells (Fig. 5A). Most of the extra 59Fe taken up by IRE-transfected cells was found in the basolateral medium (Fig. 5B). Thus, IRE-transfected cells behaved like Fe-deficient cells, with elevated apical iron uptake and efficient transfer of iron to the basolateral medium (Tapia et al., 1996).


Figure 5. 59Fe uptake by IRE-transfected cells. A: kinetics of hepithelial 59Fe transport by control and IRE-transfected cells. Insert grown pcDNA3 or pcDNA3-IRE, Caco-2 cels, were incubated with 10 µM 59Fe-NTA in the apical medium, and the amount of radioactivity found in the basolateral medium was determined as a function of the incubation time. The rates of 59Fe transport to the basolateral meduim, estimated from the slopes of the curves, were 6.0 ± 0.3 pmol x h-1, and 13.7 ± 0.8 pmol x h-1, for cells transfected with pcDNA3 and pcDNA3-IRE, respectively. B: 59Fe distribution in cells and basolateral medium after incubation for 3 h with 10 µM 59Fe-NTA in the apical medium. Data shown is the mean ± SD of 3 independent experiments.

The increased apical iron uptake could be due to increased expression of a putative apical iron transporter or to increased activity of the transporter. In the latter case, it is possible that the high levels of ferritin and the low levels of TfR induced by IRE overexpression resulted in low levels of the regulatory iron pool, which in turn should result in the shifting of the chemical equilibrium toward the entry of iron during apical iron uptake. Therefore we determined the regulatory, or labile, iron pool of cells subjected to varied manipulations (Fig. 6). While wild type and pcDNA3-transfected cells presented a sizable labile iron pool (Figs. 6A and 6D), cells transfected with pcDNA3-IRE had a very small iron labile pool (Fig. 6E). The labile iron pool was diminished in cells pre-incubated in low-iron medium (Fig. 6B), and was increased in cells preincubated in high-iron medium (Fig. 6C). Mean values, expressed in arbitrary fluorescence units for three independent determinations were 196.7, 329.3, 70.0, 180.7 and 21.0, for normal, high Fe, low Fe, pcDNA3 transfected, and pcDNA3-IRE transfected cells, respectively.


Figure 6. Determination of the labile iron pool. Wild-type Caco-2 cells (A, B), Caco-2 cells transfected with pcDNA3 (C), or cells transfected with pcDNA3-IRE (D), were grown in glass cover-slips for 10 days in DMEM, 10 % FBS. The labile iron pool was then measured either directly (A, C, and D) or after incubation for 2.5 h in an iron-poor medium (B). SIH is a membrane-permeant iron chelator that takes iron from the calcein-iron chelate thus increasing calcein fluorescence. Therefore, the Ievel of the cellular labile iron pool is directly proportional to the increase in SlH-induced calcein fluorescence. Values for fluorescence change after the addition of SIH were (mean ± SD of three determinations): A: 190 ± 16; B: 70 ± 15; C: 124 ± 28; and D: 22 ± 16.

DISCUSSION

The cellular level of iron is regulated by the activity of proteins known as iron regulatory proteins, or IRPs. IRPs bind to hairpin motifs present in the mRNA of several key proteins of iron metabolism, such as ferritin and TfR, modulating their translation. Since both the mRNA binding activity of lRPs and iron uptake by intestinal epithelia cells are regulated by the cellular level of Fe, in this study we investigated whether the activity of the IRE/IRP system regulates intestinal iron absorption. To that end, we determined the relationship between intracellular iron levels and the activity of the IRE/IRP system. We then overexpressed IRE in Caco-2 cells, reasoning that by binding to available IRP, excess IRE should functionally decrease or eliminate IRP mRNA binding activity. This approach was preferred to the expression of dominant negative IRP, since intestinal cells have both IRP I and IRP2, so cells should be transfected with both IRP 1 and IRP2 dominant negative genes to abrogate IRP activity.

We found that Caco-2 cells had an active IRE/IRP system with a peculiar characteristic: it presented a basal IRP2 activity that was unresponsive to changes in intracellular iron. This basal activity ensured basal levels of TfRs. The question arises then, with respect to the relevance of maintaining basal levels of TfRs. As the TfR is part of the system by which intestinal cells sense body iron levels, we hypothesize that the basal IRP activity is necessary to maintain the body iron sensor system operative even at high body iron levels.

Regardless of the extracellular iron concentration present during cell culture, cells overexpressing IRE presented constitutively high levels of intracellular Fe, probably the result of high ferritin levels (Fig. 2). But, contrary to the expected, apical 59Fe uptake was higher in IRE cells than in control cells (Fig. 3). Since the passage of iron from the lumen of the intestine into the enterocyte is mediated by one or more membrane iron transporters, the increased iron uptake observed in IRE cells could be due to increased expression of the transporters or to their increased activity. Two mammalian membrane iron transporters have been cloned, Nramp2, also named DCT I, (Gunshin et al., 1997; Fleming et al., 1997) and SFT (Gutierrez et al., 1997). DCT I is an electrogenic cation-H+ co-transporter with high levels of transcripts in kidney and the microvilli region of intestine (Gunshin et al., 1997). STF is an iron transporter identified by expression cloning of a K562 library, found in perinuclear vacuolar structures resembling recycling endosomes (Gutierrez et al., 1997). The tissue and cellular locations of Nramp2 and SFT indicate that the primary function of STF may be the acquisition of Tf-derived iron through the endocytosis process, while the function of Nramp2 may be the apical transport of iron by intestinal epithelia cells. While the mRNA of STF does not have an IRE motif, alternative splicing of Nramp2 produces a Nramp2 without IRE and a Nramp2 with one IRE motif in its 3'untranslated region (Lee et al., 1998). 11, by analogy with TfR mRNA, the binding of IRP stabilizes Nramp2 (IRE) mRNA, then decreased translation of this mRNA should be expected in Caco-2 IRE cells.

The results found in this work indicate that apical iron transport activity was increased in Caco-2 cells overexpressing the ferritin IRE. Therefore, any effect of IRE overexpression on decreasing Nramp2 (IRE) mass was overcome by its effects on other component(s) involved in the iron absorption process. Indeed, the high levels of ferritin and the low levels of Tf R induced by IRE overexpression resulted in very low levels of the labile iron pool. This decrease should result in a favorable chemical gradient for the entry of iron during apical iron uptake. This favorable gradient may underlie the observed increase in apical iron uptake by Caco-2-lRE cells. Nevertheless, the possible participation of some still undescribed IREcontaining element that might effect a negative control on iron absorption can not to be discarded.

In summary, Caco-2 cells overexpressing IRE presented elevated levels of ferritin and diminished levels of TfR, as would be expected if excess IRE bound to active IRP and eliminated IRP control of ferritin and TfR synthesis. Moreover, IREoverexpressing cells presented constitutively low levels of the labile iron pool, as well as high rates of apical iron uptake and transepithelial iron transport. Thus, the present results indicate that the IRE/IRP system regulates intestinal iron absorption through the regulation of the labile iron pool.

ACKNOWLEDGMENTS

This work was supported by Fondo Nacional de Ciencia y Tecnología grants 1970465 and 2970003, by Mideplan project number P99031 granted to the Instituto Milenio de Estudios Avanzados en Biología Celular y Biotecnología, and by a Cátedra Presidencial en Ciencia to MTN. We are grateful to Dr. Prem Ponka lor providing SIH and to Dr. Ioav Cabantchick for advice on the use of the LIP measurement technique.

Corresponding author: Dr. Marco T. Nuñez. Departamento de Biología, Facultad de Ciencias, Universidad de Chile. Casilla 653, Santiago, Chile. Fax: 562 271-2983. E-mail: maunez@uchile.cl

Received: December 10, 1999. Accepted: December 10, 1999

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