DMT1 (IRE) expression in intestinal and erythroid cells is regulated by peripheral benzodiazepine receptor-associated protein 7.

The divalent metal transporter 1 (DMT1) is essential for cellular uptake of iron, mediating iron absorption across the duodenal brush border membrane. We have previously shown that with iron feeding DMT1 in the brush border membrane undergoes endocytosis into the subapical compartment of enterocytes. To understand the mechanisms of iron-induced endocytosis of DMT1, we used the yeast two-hybrid system to find proteins that interact with DMT1 and isolated from a rat duodenal cDNA library a protein that interacts specifically with the IRE containing isoform of DMT1 {DMT1 [iron-responsive element (IRE)]}. The protein (Genbank AY336075) is 97.5% identical with peripheral benzodiazepine receptor-associated protein 7 (PAP7), a protein that interacts with the peripheral benzodiazepine receptor. PAP7 is ubiquitously expressed in the rat and in multiple cell lines with consensus sequences including a nuclear localization signal and a Golgi dynamic domain. PAP7, expressed on the brush border of rat duodenum, copurified with DMT1 in brush border membrane vesicles, and following iron feeding, was internalized in parallel with the internalization of DMT1. To determine if PAP7 plays a role in cellular iron metabolism, we downregulated PAP7 expression in K562 cells with small interfering RNA. Following the decrease in PAP7 protein, DMT1 (IRE) protein but not mRNA was significantly downregulated but without effect on DMT1 (non-IRE), transferin (Tf)R1, or ferritin expression. Lowered levels of PAP7 resulted also in decreased cell proliferation and G(1) cell cycle arrest. These data are consistent with PAP7 interacting with DMT1 (IRE) and regulating DMT1 (IRE) expression in K562 cells by modulating expression of DMT1 (IRE) protein.

Interactions between ferroportin and hephaestin in rat enterocytes are reduced after iron ingestion.

BACKGROUND & AIMS: Ferroportin (Fpn) is a multiple transmembrane protein required for iron export into the systemic circulation, in cooperation with hephaestin (Heph). Despite the importance of Fpn in iron transport, there is controversy about its topology and functional state upon interaction with Heph. METHODS: The topology of Fpn was determined using monospecific antisera against its different epitopes, in sheets of cells from duodenum that were or were not permeabilized with detergent. Immunoprecipitation and blue native polyacrylamide gel electrophoresis, followed by immunoblot analysis, were used to determine the extent of interactions between Fpn and Heph. Antisera against the intracellular, C-termini of divalent metal transporter (Dmt1) and Heph served as controls. RESULTS: Immunofluorescence analysis with antisera against amino acids 172-193 of Fpn (anti-Fpn 172) detected Fpn only in permeabilized cells, whereas anti-Fpn 232 (amino acids 232-249), anti-Fpn 370 (amino acids 370-420), and anti-Fpn C (the C-terminus) detected Fpn in nonpermeabilized and permeabilized cells. Immunoprecipitation studies showed that Fpn and Heph coprecipitated with either anti-Fpn or anti-Heph. Blue native polyacrylamide gel electrophoresis studies revealed that a fraction of Fpn comigrates with Heph; the apparent interaction decreases after iron ingestion. CONCLUSIONS: Studies with antisera to different epitopes of Fpn indicate that the topology of Fpn is consistent with an 11-transmembrane model, with the C-terminus exposed on the cell surface. Reduced interactions between Fpn and Heph after iron ingestion indicate that this is a regulatory mechanism for limiting further iron absorption.

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine.

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium.

Intestinal iron absorption involves proteins located in the brush border membrane (BBM), cytoplasm, and basolateral membrane (BLM) of duodenal enterocytes. Ferroportin 1 (FPN1) and hephaestin (Heph) are necessary for transport of iron out of enterocytes, but it is not known whether these two proteins interact during iron absorption. We first examined colocalization of the proteins by cotransfection of HEK293 cells with pDsRed-FPN1 with pEmGFP-Heph or with the COOH-terminal truncated pEmGFP-HephDelta43 or -HephDelta685 and found that FPN1 and Heph with or without the COOH terminus colocalized. In rat duodenal enterocytes, within 1 h of iron feeding prominent migration of FPN1 from the apical subterminal zone to the basal subnuclear zone of the BLM occurred and increased to at least 4 h after feeding. Heph exhibited a similar though less prominent migration after iron ingestion. Analysis using rat duodenal epithelial cell sheets demonstrated that 1) by velocity sedimentation ultracentrifugation, FPN1 and Heph occupied vesicles of different sizes prior to iron feeding and migrated to similar fractions 1 h after iron feeding; 2) by blue native/SDS-PAGE, FPN1, and Heph interacted to form two complexes, one containing dimeric FPN1 and intact Heph and the other consisting of monomeric FPN1 and a Heph fragment; and 3) by immunoprecipitation, anti-Heph or anti-FPN1 antiserum coimmunoprecipitated FPN1 and Heph. Thus the data indicate that FPN1 and Heph migrate and interact during iron feeding and suggest that dimeric FPN1 is associated with intact Heph.

Iron Imports. V. Transport of iron through the intestinal epithelium.

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.

Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat.

Hepcidin has been implicated as the iron stores regulator: a hepatic signaling molecule that regulates intestinal iron absorption by undefined mechanisms. The possibility that hepcidin regulates the expression of ferroportin 1 (FPT1), the basolateral iron transporter, was examined in rats after administration of LPS, an iron chelator, or His-tagged recombinant hepcidin (His-rHepc). In the liver, LPS stimulated a biphasic increase of hepcidin mRNA with peaks of mRNA at 6 and 36 h. Concurrently, hepatic FPT1 mRNA expression decreased to minimal level at 6 h and then increased with a peak at 24-36 h. LPS also induced biphasic changes in intestinal FPT1 mRNA expression, with decreased levels at 6 h and increased expression at 48 h. Whereas the initial decrease of FPT1 coincides with an LPS-induced decrease in serum iron, both intestinal and hepatic FPT1 expression recovered, whereas serum iron concentration continued to decrease for at least 24 h. Dietary iron ingestion increased intestinal ferritin protein production but did not reduce intestinal FPT1 mRNA expression. The iron chelator pyrrolidinedithiocarbamate (PDTC) stimulated hepatic hepcidin without suppressing intestinal FPT1 expression. In PDTC-treated rats, LPS stimulated no additional hepatic hepcidin expression but did increase intestinal FPT1 expression. Administration of HisrHepc induced significant reduction of intestinal FPT1 expression. Taken together, these data suggest that hepcidin mediates LPS-induced downregulation of intestinal FPT1 expression and that the hepcidin signaling pathway involves a PDTC-sensitive step.

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium.

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.