ARIEL ORELLANA,l. MARCO A. GARATE,l AND MARCO TULIO NUT;TEZl ... Marco A. G&rate, and Marco Tulio Ntifiez ..... Alvarez-Hernandez,. X., G. M. Nichols,.
Intracellular IRP activity
iron regulates iron absorption and in intestinal epithelial (Caco-2) cells
MIGUEL ARREDOND0,1y2 ARIEL ORELLANA,l MARCO A. GARATE,l AND MARCO TULIO NUT;TEZl IDepartamen to de Biologia, Facultad de Ciencias, and 21nstituto de Nutricidn y Tecnologia de 10s Alimentos, Universidad de Chile, Casilla 653, Santiago 1, Chile Arredondo, Miguel, Ariel Orellana, Marco A. G&rate, and Marco Tulio Ntifiez. Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G275-G280, 1997. -In vertebrates, body Fe homeostasis is maintained through the regulation of its intestinal absorption. In addition, because Fe is both essential and toxic, intracellular Fe levels are tightly regulated. Consequently, intestinal epithelial cells are in the unique position of being responsible simultaneously for the regulation of body Fe absorption and the regulation of their intracellular Fe levels to remain viable. We tested the hypothesis that the regulation of transepithelial Fe transport and the regulation of intracellular Fe levels are sensitive to a common effector. To this end, we used a recently developed protocol to obtain cultured intestinal epithelial cells with defined intracellular Fe concentrations. In these cells we tested Fe absorption and Fe regulatory protein (IRP) activities. We found that transepithelial Fe transport was inversely related to 20-200 PM intracellular Fe and that Caco-2 cells expressed Fe regulatory protein-l and Fe regulatory protein-2 activities. Fe regulatory protein-l activity, Fe regulatory protein-2 mass, transferrin receptor density, and ferritin levels were regulated by intracellular Fe in the same range (20-200 PM) that affected transepithelial Fe transport. These results suggest that a common Fe-responsive factor regulates both intracellular Fe levels and Fe absorption by Caco-2 cells. iron homeostasis; ferritin; tory protein; iron transport
transferrin
receptor;
iron regula-
DO NOT HAVE regulated excretory mechanisms for Fe. Consequently, their body Fe homeostasis is maintained mainly through the regulation of intestinal Fe absorption. Intestinal epithelial cells respond to a fall in body Fe stores by increasing the absorption of dietary Fe, so the extent of Fe transport through the intestinal epithelium is inversely related to the content of body Fe stores (4,9,20; reviewed in Ref. 6). Knowledge of the molecular mechanisms involved in the regulation of transepithelial Fe transport remains elusive, in part because of the heterogeneity in age and Fe content of intestinal cells. Intestinal epithelial cells proliferate in the bases of the intestinal villi, migrate to the tips of the villi, and then slough off into the intestinal lumen. In humans this process lasts -3 days. Because during this time the cells continuously acquire Fe, older cells contain more Fe than younger cel1.s(3). This heterogeneity in cell age and cell Fe content hinders the establishment of experimental conditions suitable to study in vivo the expression, or activity, of proteins invol.ved in the regulation of Fe absorption. Therefore, cultured human cell lines that undergo MAMMALS
0193~1857/97
$5.00
Copyright
o 1997
spontaneous differentiation to enterocytes are used, because they represent cell populations homogeneous in age. In particular, Caco-2 cells have been described as an excellent in vitro model of human enterocytes (1, 14). Caco-2 cells grown on bicameral inserts exhibit both apical Fe uptake, responsive to the intracellular content of Fe (1,27), and transferrin-mediated basolatera1 Fe uptake (23). In addition, Caco-2 cells reduce Fe3+ to Fe2+ in the apical medium, and this reduction correlates with increased Fe uptake (11, 22). Caco-2 cells cultured in high-Fe medium absorb less Fe than cells cultured in low-Fe medium (1). Recently, using a technique to grow Caco-2 cells to well-defined intracellular Fe levels, we found that the mechanisms responsible for the regulation of Fe absorption seem to be responsive to changes in intracellular Fe (27). Fe regulatory proteins (IRPs) are cytosolic proteins that bind to structural elements, named Fe-responsive elements (IREs), present in the untranslated region of mRNAs that code for ferritin, the transferrin receptor, and aminolevulinate synthase (2, 19, 21, 26). On the basis of the structure of mitochondrial aconitase, IRP-1 (relative mobility 98 kDa) has been proposed to possess four domains, in which domains 1, 2, and 3 are connected to domain 4 through a hinge region (reviewed in Ref. 18). Low levels of intracellular Fe cause IRP-1 to bind to and stabilize transferrin receptor mRNA and to bind to ferritin mRNA, diminishing its translation. The mRNA binding activity of IRP-1 is activated by 2-mercaptoethanol (2-ME) and inhibited by N-ethylmaleimide (17). IRP-2 (relative mobility 104-105 kDa) has 65% homology with IRP-1; it has a 79-amino acid insertion in domain 1 (24), and it is present in normal brain and intestinal tissue (13). IRP-2 undergoes Feinduced proteolytic degradation, and its activity is not enhanced by 2-ME (10, 15, 25). The activities of IRP-1 and IRP-2 respond to cellular Fe but through different mechanisms: IRP-1 activity is regulated by a posttranslational mechanism (21, 26), whereas IRP-2 inactivation by Fe reflects IRP-2 protein degradation (10, 13, 15,25). Fe homeostasis in duodenal cells is rather unique, because these cells must regulate their intracellular Fe levels and transcellular Fe transport. Few studies are available on the regulation of intracellular Fe levels in intestinal epithelial cells (7, 24, 28). IRP activity was normal in individuals with hemochromatosis (7). Similarly, a decrease in ferritin expression takes place in the duodenum from individuals with idiopathic hemochromatosis or Fe deficiency anemia, an indication of active IRP (24). In contrast, IRP activity does not respond to Fe loading in rat duodenum (28). the American
Physiological
Society
G275
G276
INTESTINAL
IRON
ABSORPTION
Because intracellular Fe is involved in regulating IRP activity and Fe absorption, we investigated in Caco-2 cells the range of intracellular Fe concentrations in which both functions respond. We found that Fe absorption, ferritin levels, transferrin receptor density, and IRP activity were regulated within the same 20-200 JLM total intracellular Fe, with a K0 5 (operational constant that indicates the concentration of intracellular Fe at which the parameter under study has one-half of its maximal activity) for all these processes of -50 JLM Fe. We propose that intestinal Fe absorption and the activity of the IRP system are under the control of a common Fe-sensitive regulator that operates at 20-200 PM intracellular Fe. EXPERIMENTAL
PROCEDURES
Equilibrium loading of Caco-2 cells with 55Fe. The procedure to obtain cells with known concentrations of Fe is described elsewhere (27). Briefly, Caco-2 cells (American Type Culture Collection, Rockville, MD) were seeded at I X lo5 cells/25cm2 flask and incubated for 1 wk in low-Fe medium (GIBCO Laboratories, Grand Island, NY) and 10% low-Fe serum (co.1 PM Fe) (I), supplemented with various amounts of Fe3+ as the complex 55FeC13-sodium nitrilotriacetate (55FeNTA, 1:2 molar ratio). During this period the cells reached confluence, with 2-4 X lo6 cells/25cm2 flask. The cells were trypsinized and seeded at a density of I X lo5 cells/flask and cultured as described above. This procedure was repeated once. The cells obtained from the second trypsinization were plated onto 0.33-cm2 polycarbonate inserts (Transwells, Costar, Cambridge, MA) for Fe transport experiments or in plastic flasks and were cultured in media as described above for 14 days, with change of media every 3-4 days. The formation of a cell monolayer in the inserts was monitored by measuring the transepithelial electrical resistance with an EVOM epithelial voltohmmeter (World Precision Instruments, Sarasota, FL). The total intracellular Fe concentration was estimated from the specific radioactivity of the 55Fe in the medium and a cell volume of 1 ,u1/0.33-cm2 insert (23). The values represent total intracellular Fe. With the activation of aconitase by Fe2+ as the reference, the free intracellular Fe concentration should be 1O-1o-1O-g M (16). CeZZ extracts. To prepare cell extracts, cells were treated with lysis buffer containing 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.5, 3 mM MgC12, 40 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 10 pglml leupeptin, 0.5 pg/ml aprotinin, 0.7 pglml pepstatin A, 5% glycerol, 1 mM dithiothreitol, and 0.5% Triton X-100 at 50 pi/l X lo6 cells. The mixture was incubated for 15 min on ice and centrifuged for 10 min at 10,000 g, The supernatant was stored at -70°C and used for the determination of ferritin, transferrin receptor density, IRP activity, and IRP immunodetection. 5gFe fluxes. For 5gFe flux experiments we used 55Fecontaining cells grown on 0.33-cm2 inserts for 14 days as described above. At this time there were, on average, 220,000 cells/insert, and the transepithelial electrical resistance was 250-300 S1cm2. To ensure integrity of the cell barrier, we measured the transepithelial electrical resistance of the inserts at the beginning and end of each experiment and discarded all inserts with transepithelial electrical resistance ~220 ficm2 at the end of the experiment. Cells were incubated at 37°C for 30-120 min with 1 PM Fe3+, as the 5gFe-NTA complex, added to the apical medium. The incubation medium was low-Fe Dulbecco’s modified Eagle’s medium.
AND
IRP ACTIVITY
Fe uptake was stopped by washing the inserts three times with ice-cold saline supplemented with 1 mM EDTA. Transepithelial Fe transport experiments were carried out using 1 PM 5gFe-NTA in the apical medium. 55Fe and 5gFe radioactivity in the cells and in the basolateral media was measured in a dual-channel beta counter (Packard Instrument, Meriden, CT). Uptake and transepithelial transport of 5gFe were linear during the first 2 h of incubation. Uptake was expressed as the rates (mol 5gFe h-l insert-l) estimated by linear regression fitting of the data. Immunodetection of IRP. IRP mass in lysates from Caco-2 cells with different concentrations of Fe was estimated by Western blotting (12). Cell extract protein (10 pg) was separated in 7.5% sodium dodecyl sulfate-polyacrylamide gels. The proteins were then transferred to nitrocellulose and reacted with primary antibody. Primary antibody was a custom-made rabbit polyclonal antibody prepared against the synthetic peptide NSYGSRRGNDAVMARC (Bios-Chile, Santiago, Chile), as described previously (5). The NSYGSRRGNDAVMARC sequence is part of domain 4 in IRP-1 and IRP-2 (13). The secondary antibody was peroxidase-labeled goat anti-rabbit immunoglobulin G (Sigma Chemical, Saint Louis, MO). The membranes were developed using the enhanced chemiluminescence Western blotting detection kit (Amersham, Arlington Heights, IL) and were exposed to autoradiographic film. The relative intensity of the bands was determined by densitometric analysis using the SigmaScan program (Jandel Scientific, San Rafael, CA) after subtraction of background values obtained for each lane. Band-shift assay of IRP IRP activity was determined by the band-shift assay described by Leibold and Munro (19). 32P-labeled IRE was prepared by in vitro transcription of pSPT-fer (21) (the kind gift of Drs. Rogerio Meneginni and Lukas Khun) using 32P-labeled uridine 5’-triphosphate (Dupont NEN, Boston, MA). 32P-labeled IRE was incubated with Caco-2 cell extracts for 30 min at room temperature. When indicated, cell extracts were preincubated with 2% 2-ME for IO min at room temperature. One unit per assay of ribonuclease Tl was added, and the incubation was continued for 10 min. Heparin (5 mg/ml) was then added, and the incubation was continued for 10 min. The mixture was loaded onto 5% nondenaturant 16 X 18-cm polyacrylamide gels and electrophoresed at 200 V The gel was dried, and its 32P radioactivity was detected by autoradiography. The relative intensity of the bands was determined using the SigmaScan program. In some experiments the bands were excised, and their radioactivity was determined in a radioactivity counter. Good correlation was always observed between these two methods of quantification. Determination of intracellular ferritin levels. Intracellular levels of ferritin were determined in extracts from Caco-2 cells containing different concentrations of Fe, using a sandwich enzyme-linked immunosorbent assay, as described previously (8). Polyclonal rabbit anti-human ferritin and peroxidase-labeled rabbit anti-human ferritin antibodies were purchased from Dako (Carpinteria, CA). Measurement of transferrin receptor density. Transferrin receptors were determined in cell extracts by an enzymelinked immunosorbent assay (12), using OKT9 antitransferrin receptor monoclonal antibody as primary antibody and peroxidase-labeled goat anti-mouse immunoglobulin G (Sigma Chemical) as secondary antibody. Data analysis. In all the experiments, variables were tested in triplicate wells, and the experiments were repeated three to five times. Variability between experiments was ~10%. Figures l-4 show representative experiments. Curve l
l
INTESTINAL
fitting was done using the GraphPad Pad Software, San Diego, CA).
Prism
IRON
program
ABSORPTION
AND
G277
IRP ACTIVITY
(Graph-
RESULTS
Relationship between intracellular 55Fe levels and Fe uptake. Caco-2 cells were grown in media with various 55Fe concentrations to obtain cells with different levels of 55Fe, as described previously (27). In these cells the rate of apical-to-cell 5gFeuptake was measured (Fig. 1). 5gFe apical uptake decreased from 7.1 pmol/h with 20 JLM intracellular Fe to -4.7 and 3.1 pmol/h with 80 and 300 PM intracellular Fe, respectively. Further increases in the concentration of intracellular Fe up to 680 PM did not produce further changes in apical Fe uptake (not shown). The Ko5 for these changes was 46.2 t 5.1 (SD) PM Fe (n = 5). These results indicate that apical 5gFe uptake changed only in a defined range of intracellular Fe concentration. Transepithelial 5gFetransport also responded to intracellular Fe levels. Cells with 30 PM intracellular Fe, the lowest concentration studied, displayed the highest rate (6.4 pmol/h) of apical-to-basolateral 5gFe flux. Cells containing higher intracellular Fe had lower transport rates. Thus, in cells with 80 and 300 PM Fe, the apical-to-basolateral 5gFe transport rate decreased markedly to 0.8 and 0.6 pmol/h, respectively (Fig. 1). Kom5 for transepithelial transport was 15.2 t 2.1 PM Fe. We also determined the effect of varying intracellular Fe concentration on cell-to-basolateral 55Fe fluxes. At 580 PM intracellular Fe the contribution of cellular 55Fe to the overall cell-to-basal Fe flux was relatively small and was lower than the apical-to-basal 5gFe flux. At >300 PM cellular 55Fe, however, 55Fe from intracel-
apical
to cells
apical cells
Total
to basal to basal
intracellular
59Fe flux 59Fe flux 5sFe flux
Fe, pM
Fig. 1. Transepithelial 5gFe transport as a function Fe concentration. Insert-grown cells were equilibrated intracellular concentrations indicated, and apical-to-cell to-basal 5gFe fluxes were determined after addition sodium nitrilotriacetate to apical medium and further of 5gFe radioactivity in cells and in basolateral medium. 55Fe fluxes were obtained from 55Fe radioactivity medium at different times of incubation.
of intracellular with 55Fe to and apicalof 1 JLM 5gFedetermination Cell-to-basal in basolateral
I
50
I
100
Intracellular Fig. 2. function dishes cated. mined
I
150
200
Fe, pM
Transferrin receptor (RTf) density and ferritin (Fn) levels as a of intracellular Fe levels. Cells were cultured in plastic to equilibration with intracellular 55Fe concentrations indiTransferrin receptor density and ferritin levels were deterfrom cell extracts.
lular compartments became the predominant species exchanging between the cell and the basolateral medium (Fig. 1). These combined results confirm previous findings (27) establishing that Fe absorption is regulated by the cellular Fe concentration. Furthermore, at any given intracellular Fe concentration the rate of transepithelial Fe transport was always smaller than the rate of apical Fe uptake, indicating that there is also intracellular regulation of Fe absorption. Relationship between intracellular Fe content, transferrin receptor density, and ferritin levels. Transferrin receptor density decreased in cells with increasing Fe levels from 24 to 188 PM (Fig. 2). Curve fitting of the data indicated a& .5 of 56.1 t 7.5 (SD) PM Fe (n = 4). In contrast, ferritin levels increased in cells with increasing Fe concentrations, with a steeper increment at 24-114 PM Fe (Fig. 2). The K0 5 for ferritin increase was 52.4 t 6.1 PM Fe (n = 4). ’ IRP activity and intracellular Fe levels in Caco-2 cells. The decrease in transferrin receptor density and the concomitant increase in ferritin levels as a function of intracellular Fe suggested that these responses were mediated by IRP. So we searched for IRP activity in Caco-2 cells with various intracellular Fe concentrations. Determination of IRP activity in Caco-2 cells by band-shift assay revealed two bands of activity that we named Pl and P2 (Fig. 3A). The activity of PI varied inversely with cellular Fe content, being more distinctive at 28-203 PM Fe (Fig. 3B). The K0 5 for the process Fe (n = 3). In was 43.4 t 8.6 (SD) PM intracellular contrast, the activity of P2 decreased a modest 20% between 28 and 203 JLM Fe (Fig. 3B). Treatment of extracts with 2-ME increased Pl, but not P2, activity (Fig. 3A, cf. lanes 3-7 with lanes 8-12). On the basis of 2-ME and Fe sensitivity, Pl and P2 can be ascribed to
G278
INTESTINAL
IRON
ABSORPTION
A 1 2
3 4
5 6
7
8 9 10 11 12
/ i
I Fe, PM:
2h4Jz: -
50 28 50 105203685
28 50 105203685
-
+
-
-
-
-
-
+
+
+
-Pl - P2
+
AND
IRP ACTIVITY
from cells containing 16-80 ,z& intracellular Fe, decreasing to -50% in extracts from cells containing 80-196 PM Fe (Fig. 4B). In contrast, the intensity of P98 did not change at 16-196 PM Fe (Fig. 4B). Taken together, these results indicate that the total IRP mass (IRP-1 + IRP-2) decreased a modest 25% at 16-196 PM Fe. On the basis of their molecular weights and sensitivity to cell Fe levels, P98 and P105 can be tentatively ascribed to IRP-1 and IRP-2, respectively (13, 15, 25, 26). DISCUSSION
B P2 -”
80
f x
60
A
Intestinal epithelial cells regulate the transcellular flux of Fe from the lumen of the intestine to the circulating plasma. We found that in Caco-2 cells changes in apical Fe uptake, transepithelial Fe transport, transferrin receptor density, and ferritin levels were restricted to cells with a limited range of cellular Fe concentrations. Increments in the intracellular Fe
A
200
2
3
4
5
6
7
8
-
16
26
59
80
105 157 196
600
400
Intracellular
1
Fe, pM
Fig. 3. Determination of Fe regulatory protein (IRP) activity in extracts from cells with various Fe concentrations. A: band-shift assay of IRP activity. Caco-2 cell extracts were incubated with 32P-labeled Fe-responsive element (IRE) without (lanes 3-7) or with (lanes 8-12) 2% P-mercaptoethanol. Mixture was then separated in nondenaturant 5% polyacrylamide gel, and dried gel was autoradiographed. Lane 1, 32P-IRE without cell extract; lane 2, cell extract without s2P-IRE; lanes 3 and 8, 28 PM internal Fe; lanes 4 and 9, 50 PM internal Fe; lanes 5 and 10, 105 ,uM internal Fe; lanes 6 and 11, 203 PM internal Fe; lanes 7 and 12,685 PM internal Fe. B: densitometric analysis of bands in lanes 3-7. Band intensity was estimated as described in EXPERIMENTAL PROCEDURES. Intensity of Pl band in lane 3 was arbitrarily set at 100, and intensities of other bands are relative to Pl.
Fe,pM
IRP-1 and IRP-2, respectively (10, 13, 27). Thus it appears that Caco-2 cells have both IRP-1 and IRP-2 activities. In cells with low Fe concentrations (e.g., 28 PM) IRP-1 is the prevalent activity, whereas in cells with higher Fe concentrations (>203 PM) IRP activity is constitutive, mostly in the form of IRP-2. IRP immunoreactivity
and intracellular
Fe levels. We
next tested whether the mass of IRP is regulated by Fe in Caco-2 cells. Two bands of 105 and 98 kDa (named P105 and P98) were evident when extracts derived from cells with various intracellular Fe levels were reacted with anti-IRP antibody (Fig. 4A). In control experiments the immunoreactivity of these bands was completely abolished when the IRP peptide NSYGSRRGNDAVMARC (see EXPERIMENTAL PROCEDURES) was added to the cell extracts or when preimmune serum was used as primary antibody (not shown). The immunoreactivity of P105 remained constant in extracts
I
50
100
Intracellular
150
200
Fe, pM
Fig. 4. Immunodetection of IRP in extracts from Caco-2 cells with various Fe concentrations. A: Western blotting. Caco-2 extracts (10 pg protein) were electrophoresed in a 10% polyacrylamide-SDS gel and later transferred to nitrocellulose membrane. IRPs were detected with specific antibodies. Lane 1, no extract; lanes 2-8, extracts from cells with 16, 26, 59, 80, 105, 157, and 196 PM Fe, respectively. B: densitometric analysis of immunodetected bands. Band intensity was estimated as described in EXPERIMENTAL PROCEDURES. Intensity of P105 in lane 2 was arbitrarily set at 100, and intensities of other bands are relative to P105.
INTESTINAL
IRON
ABSORPTION
concentration from 20 to -200 JLM resulted in a decrease in transepithelial Fe flux and density of transferrin receptors and an increase in ferritin levels. The present results also indicate that not all the Fe incorporated from the apical medium was transported to the basolateral side, an indication that Fe absorption is also regulated by intracellular factors. Because increasing cellular Fe induced increased ferritin levels, the intracellular regulation may be sustained by ferritin. The increase in ferritin levels as a function of cellular Fe showed a plateau at -28 nM ferritin (Fig. 2). Considering a maximal Fe binding capacity of 4,500 atoms per molecule, ferritin should saturate at -130 PM intracellular Fe. Nevertheless, there was an effective intracellular regulation of transepithelial Fe transport up to 300 PM Fe, expressed by the difference between apical-to-cell and apical-to-basolateral 5gFe flux in Fig. 1. It follows that, at > 130 JLM intracellular Fe, factors other than ferritin may be involved in the regulation of transepithelial Fe transport. Immunodetection of IRP in Caco-2 cells revealed the presence of two bands tentatively ascribed to IRP-1 and IRP-2. IRP-1 mass did not change in cells with increasing intracellular Fe concentrations, whereas the mass of IRP-2 decreased -50% in cells with SO-196 PM Fe. Therefore, Caco-2 cells, like other cell types (10, 25), have Fe-mediated degradation of IRP-2 but not of IRP-1. IRP-1 activity markedly diminished in cells with 17-203 PM intracellular Fe, whereas IRP-2 activity decreased only -20% in the same range. It is not clear why the decrease in IRP-2 activity was not larger, since a 50% decrease in IRP-2 mass was evident by Western blotting. It may be that Caco-2 cells have a heterogeneous IRP-2 pool, with a fraction of it less responsive to Fe-induced degradation but with higher affinity for IRE, which would lead to a sustained activity. From the physiological standpoint, this IRP-2 activity refractory to Fe-induced regulation results in the expression of a basal level of transferrin receptors while limiting the cellular levels of ferritin. The finding that overall IRP activity preferentially responded to a limited range of cellular Fe concentration provides a possible explanation to seemingly contradictory results on IRP activity in intestinal cells (7, 24, 28). In patients with anemia and hemochromatosis, conditions in which low intracellular Fe levels are expected, translation of duodenal ferritin and transferrin receptor mRNAs was regulated in concert, suggesting that an active IRP system was operating (7,24). On the other hand, it has been reported that duodenal IRP activity did not respond to Fe loading in rats (28). It is possible that, in the case of anemic patients, intracellular Fe levels were ~200 JLM, a range of Fe concentration where changes in IRP activity mirror changes in Fe content. Fe loading in rats could have shifted intracellular Fe levels from an unknown basal level to ~200 PM. If the basal Fe was 2100 ,uM, little change in IRP activity should be expected on Fe loading. In summary, we found that Caco-2 cells responded to increases in intracellular Fe up to -200 PM by reduc-
AND
IRP
G279
ACTIVITY
ing IRP-1 activity, apical Fe uptake, and transepithelial Fe transport. A low IRP activity, given by IRP-2, as well as a low activity of Fe uptake and transepithelial transport, remained at ~200 JLM intracellular Fe. These coordinated responses suggest that Fe absorption and IRP activity are under the control of a common Fe-sensitive regulator that operates in cells with ~200 PM cellular Fe. This work was financed by Fondo National de Ciencia y Tecnologia, Grant 1940568, by a Catedra Presidential to M. T. Nunez, and by a Departamento Tecnico de Investigation, Universidad de Chile grant to M. Arredondo. Address for reprint requests: M. T. Nunez, Dept. de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago-l, Chile. Received
12 December
1996;
accepted
in final
form
7 April
1997.
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G280
INTESTINAL
IRON
ABSORPTION
binding proteins, IRPl and IRP2. J. Biol. Chem. 270: 4983-4986, 1995. 18. Klausner, R. D., T. A. Rouault, and J. B. Hartford. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72: 19-28, 1993. 19. Leibold, E. A., and H. N. Munro. Cytoplasmic protein binds in vitro to a highly conserved sequence in the untranslated region of ferritin heavyand light-subunit mRNAs. Proc. Nat,!. Acad. Sci. USA 85: 2171-21751988. 20. Linder, M. C., and H. N. Munro. The mechanism of iron absorption and its regulation. Federation Proc. 36: 2017-2023,1977. 21. Mullner, E. W., B. Neupert, and L. C. Kuhn. A stem-loop in the 3’-untranslated region mediates iron-dependent regulation of the transferrin receptor mRNA stability in the cytoplasm. CeZZ 53: 815-825,1988. 22. Nufiez, M. T., X. Alvarez, M. Smith, V. Tapia, and J. Glass. Role of redox systems on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells. Am. J. PhysioZ. 267 (Cell Physiol. 36): C1582-C1588,1994. 23. Nufiez, M. T., V. Tapia, and M. Arredondo. Iron acquisition through the basolateral endocytosis of holotransferrin is inhib-
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ited by apotransferrin in intestinal epithelial (Caco-2) cells. J. Nutr. 126: 2151-2158,1996. A., E. Rocchi, G. Casalgrandi, G. Rigo, 24. Pietrangelo, A. Ferrari, M. Perini, G. Ventura, and G. Cairo. Regulation of transferrin, transferrin receptor and ferritin genes in human duodenum. GastroenteroZogy 120: 802-809,1992. 25. Samaniego, F., J. Chin, K. Iwai, T. A. Rouault, and R. D. Klausner. Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function and post-translational regulation. J. BioZ. Chem. 269: 30904-30910,1994. 26. Tang, C., J. Chin, J. B. Hartford, R. D. Klausner, and T. A. Rouault. Iron regulates the activity of the iron-responsive element binding protein without changing its rate of synthesis or degradation. J. BioZ. Chem. 267: 24466-24470,1992. 27. Tapia, V., M. Arredondo, and M. T. Ntifiez. Regulation of iron absorption by cultured intestinal epithelial (Caco-2) cell monolayers with varied Fe status. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G443-G447, 1996. 28. Ward, R. J., L. K. Kuhn, P. Kaldy, A. Florence, T. J. Peters, and R. R. Crichton. Control of cellular iron homeostasis by iron responsive elements in vivo. Eur. J. Biochem. 220: 927-931,1994.
