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HFE inhibits apical iron uptake by intestinal epithelial (Caco-2) cells¹

MIGUEL ARREDONDO^*,, PATRICIA MUÑOZ, CASILDA V. MURA and MARCO T. NÚÑEZ^*,2

^* Departamento de Biología, Facultad de Ciencias, Universidad de Chile,
Instituto de Nutrición y Tecnología de los Alimentos, and
Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, Universidad de Chile, Santiago, Chile

²Correspondence: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. E-mail: mnunez{at}uchile.cl

SPECIFIC AIMS

The physiological mechanisms by which HFE, the normal product of the HH gene, regulates intestinal iron absorption are unknown. Under the hypothesis that HFE regulates intestinal iron absorption, we characterized the effect of HFE overexpression on iron homeostasis and apical iron uptake in intestinal epithelial Caco-2 cells.

PRINCIPAL FINDINGS

1. HFE-transfected cells have decreased cellular iron levels
HFE expression was evaluated in cells transfected with the pcDNA3 vector (control cells) and cells transfected with pcDNA3-HFE (HFE cells). Densitometric analysis revealed that the HFE mass in HFE cells was fourfold larger than in the control cells (Fig. 1A ).

View larger version (18K): [in this window] [in a new window]   Figure 1. Intracellular iron content in Caco-2 cells transfected with HFE cDNA. A) Extracts from Caco-2 cells (control: transfected with the pcDNA3 vector; HFE: transfected with pcDNA3-HFE) were subjected to Western immunodetection of HFE using a polyclonal antibody raised against the carboxyl-terminal segment HFE. B) Cells were grown for 14 days in Iscove medium supplemented with 10% iron-depleted FBS and with 0.2, 1, 5, 10, or 20 µM 55Fe. The cells were then examined for 55Fe radioactivity. Plotted are the levels of intracellular 55Fe found as a function of the different concentrations of 55Fe present in the culture media.

Since the intracellular level of iron is an important regulator of Caco-2 cell iron absorption, we determined the intracellular iron content of control and HFE-transfected cells cultured with varying concentrations of iron in the culture media. Intracellular iron levels rose when control cells were incubated with increasing concentrations of ⁵⁵Fe in the culture medium (Fig. 1B ). Intracellular ⁵⁵Fe contents (pmol/mg protein) of 226 ± 27, 578 ± 47, 1724 ± 117, and 2938 ± 225 were determined when the culture media contained 1 µM; 5 µM, 10 µM and 20 µM ⁵⁵Fe, respectively. HFE cells had markedly decreased (P < 0.001) intracellular ⁵⁵Fe levels for all the extracellular iron concentration tested. Cells cultured with 1, 5, 10, 20, and 20 µM ⁵⁵Fe presented intracellular ⁵⁵Fe contents (pmol/mg protein) of 95 ± 12, 132 ± 17, 266 ± 28, and 591 ± 42, respectively. Hence, the total intracellular iron in HFE-transfected cells decreased by more than fourfold vs. those of control cells transfected with the pcDNA3 vector.

2. HFE cells have decreased activities of apical ⁵⁹Fe uptake
The decreased content of intracellular iron observed in HFE-transfected cells could be due to decreased iron uptake, and so the kinetics of apical iron uptake was investigated. Cells were grown for 2 wk in a bicameral chamber system with different concentrations of iron in the culture media and the rate of apical-to-cell ⁵⁹Fe transport was determined. Control cells with increased levels of intracellular iron showed decreased rates of apical ⁵⁹Fe uptake, as expected from normal intestinal cells that regulate iron absorption. In contrast, the uptake of ⁵⁹Fe by HFE-transfected cells was very low at all intracellular iron concentrations studied and showed no significant differences among them. Plotting the rates of apical ⁵⁹Fe transport as a function of the intracellular ⁵⁵Fe concentration produced a typical decay curve for control cells. The rates of apical ⁵⁹Fe transport by cells overexpressing HFE were considerably lower than control rates and not significantly different (P>0.5) for the four iron concentrations studied.

3. HFE cells have increased levels of DMT1
The Fe²⁺ membrane transporter DMT1 affects apical iron uptake by intestinal cells. Since a decreased rate of apical iron uptake could be the consequence of a diminished expression of DMT1, its mass was determined by Western analysis. An eightfold increase in DMT1 mass was found in HFE-overexpressing cells when compared with control cells. This observation discards the possibility that the decreased rate of apical iron uptake observed in HFE-overexpressing cells was due to the decreased expression of DMT1.

4. HFE cells have an active IRE/IRP system
The increase in DMT1 mass was to be expected if the low intracellular iron levels found in HFE-overexpressing cells were to activate the IRE/IRP system and thus DMT1-IRE expression. Accordingly, TfR levels were increased by two- to threefold in HFE-transfected Caco-2 cells when compared to control cells (data not shown). Also, levels of ferritin were decreased in HFE-transfected cells, albeit to a lower level than expected in view of the low amount of intracellular iron.

CONCLUSIONS

Body iron stores are kept relatively constant by a regulatory mechanism that limits iron uptake from the gut to precisely match iron losses. This balance is lost in hereditary hemochromatosis, a common genetic disease in humans characterized by unregulated high intestinal Fe absorption. We tested the hypothesis that HFE, the protein product of the hemochromatosis gene, should regulate intestinal iron absorption. To investigate the role of HFE in cellular iron metabolism, we studied the effects of HFE overexpression on iron homeostasis and apical iron uptake by Caco-2 cells. We found that HFE-transfected cells had a marked decrease in their intracellular iron content. Moreover, the transfected cells had a decreased rate of apical iron uptake. This decrease in apical iron uptake was not due to decreased expression of DMT1, as its mass was actually increased in HFE overexpressing cells.

There is compelling evidence to indicate that DMT1 is the transporter that mediates body iron overload in HH; although HFE knockout mice have the iron overload phenotype, the double HFE/DMT1 knockouts do not overload with iron. Taken together, these results indicate that the apical transport of iron is mediated by DMT1 and that DMT1 is a primary target of HFE action. The lower rates of apical iron uptake observed in this work confer the role of negative regulator of intestinal iron absorption on HFE and provide an explanation for the high levels of intestinal iron absorption found in hereditary hemochromatosis, where the normal function of HFE is lost.

The decrease in cell iron content observed in Caco-2 HFE cells was similar to that observed in HeLa cells overexpressing HFE, in which decreased iron uptake from diferric transferrin and the generation of an iron-deficient phenotype was reported. DMT1 could also serve as the endosomal iron transporter in Tf cycle endosomes, acting to export iron from the endosome into the cytoplasm of the cell. In the light of these results, an interpretation of the decreased transferrin-mediated iron uptake found in HeLa cells overexpressing HFE would be that the transport of iron out of the endosome is impaired because of inhibition of DMT1 activity by HFE. On the contrary, our findings do not readily agree with those recently reported in monocytes/macrophages in which HFE overexpression induced an increase in transferrin-bound iron uptake. Certainly, the different physiological roles of enterocytes and macrophages in iron handling could explain a differential regulation of DMT1 activity in these cells. The results would agree if, for example, DMT1 functions in iron export in macrophages, as suggested by Nramp2 activation.

The question arises as to how HFE could affect the activity of DMT1 in the apical membrane (Scheme 1 ). One possibility is that a fraction of HFE reaches the apical membrane where it interacts with DMT1, inhibiting its activity. Alternatively, DMT1 could interact with HFE during its destination, limiting its targeting to the apical membrane. Detailed studies of the sorting and targeting of HFE and DMT1 should clarify the locus of HFE and DMT1 interaction.

View larger version (30K): [in this window] [in a new window]   Scheme 1. A) HFE inhibits apical iron uptake by intestinal epithelial cells. B–D) Possible mechanisms of inhibition. B) DMT1 is inhibited due to a direct interaction with HFE in the apical plasma membrane sequestering DMT1 in an intracellular compartment. C) HFE interferes with the destination of DMT1 from the endoplasmic reticulum (E) to the apical plasma membrane. D) HFE is part of an unknown (U) system of signal transduction that negatively controls DMT1 activity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0578fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0578fje

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