The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels.

ATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8 mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice.

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.

Regulation of iron metabolism in Hamp (-/-) mice in response to iron-deficient diet.

BACKGROUND: Hepcidin, the liver-secreted iron regulatory peptide, maintains systemic iron homeostasis in response to several stimuli including dietary iron levels and body iron status. In addition, iron metabolism is controlled by several local regulatory mechanisms including IRP and Hif-2α activities independently of hepcidin. However, the roles of these mechanisms and their interaction particularly in hepcidin-deficient individuals are not yet fully understood. We, therefore, aimed to explore whether Hamp disruption affects iron homeostatic responses to dietary iron deficiency. METHODS: Hepcidin1 knockout (Hamp (-/-)) mice and heterozygous littermates were fed with control or iron-deficient diet for 2 weeks. The expression of iron-related genes and proteins were determined by quantitative PCR and Western blot, respectively. RESULTS: Two-week iron-deficient diet feeding in Hamp (-/-) mice did not alter serum iron but significantly reduced liver non-heme iron levels. This was also associated with increased ferroportin protein expression in the duodenum and spleen, whereas decreased expression was found in the liver. In addition, significant inductive effects of iron-deficient diet on Dcytb and DMT1 mRNA expression in the duodenum were noted with more pronounced effects in Hamp (-/-) mice compared with controls. CONCLUSIONS: Hamp (-/-) mice exhibited a more dramatic increase in the expression of iron transport machinery, which may be responsible for the unaltered serum iron levels upon iron-deficient diet feeding in these mice. Despite the lack of hepcidin, Hamp (-/-) mice can maintain a degree of iron homeostasis in response to altered dietary iron through several hepcidin-independent mechanisms.

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis.

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.

Duodenal reductase activity and spleen iron stores are reduced and erythropoiesis is abnormal in Dcytb knockout mice exposed to hypoxic conditions.

Duodenal cytochrome b (Dcytb, Cybrd1) is a ferric reductase localized in the duodenum that is highly upregulated in circumstances of increased iron absorption. To address the contribution of Dcytb to total duodenal ferric reductase activity as well as its wider role in iron metabolism, we first measured duodenal ferric reductase activity in wild-type (WT) and Dcytb knockout (Dcytb(-/-)) mice under 3 conditions known to induce gut ferric reductase: dietary iron deficiency, hypoxia, and pregnancy. Dcytb(-/-) and WT mice were randomly assigned to control (iron deficiency experiment, 48 mg/kg dietary iron; hypoxia experiment, normal atmospheric pressure; pregnancy experiment, nonpregnant animals) or treatment (iron deficiency experiment, 2-3 mg/kg dietary iron; hypoxia experiment, 53.3 kPa pressure; pregnancy experiment, d 20 of pregnancy) groups and duodenal reductase activity measured. We found no induction of ferric reductase activity in Dcytb(-/-) mice under any of these conditions, indicating there are no other inducible ferric reductases present in the duodenum. To test whether Dcytb was required for iron absorption in conditions with increased erythropoietic demand, we also measured tissue nonheme iron levels and hematological indices in WT and Dcytb(-/-) mice exposed to hypoxia. There was no evidence of gross alterations in iron absorption, hemoglobin, or total liver nonheme iron in Dcytb(-/-) mice exposed to hypoxia compared with WT mice. However, spleen nonheme iron was significantly less (6.7 ± 1.0 vs. 12.7 ± 0.9 nmol · mg tissue(-1); P < 0.01, n = 7-8) in hypoxic Dcytb(-/-) compared with hypoxic WT mice and there was evidence of impaired reticulocyte hemoglobinization with a lower reticulocyte mean corpuscular hemoglobin (276 ± 1 vs. 283 ± 2 g · L(-1); P < 0.05, n = 7-8) in normoxic Dcytb(-/-) compared with normoxic WT mice. We therefore conclude that DCYTB is the primary iron-regulated duodenal ferric reductase in the gut and that Dcytb is necessary for optimal iron metabolism.

Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling.

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism.

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.

Iron absorption in hepcidin1 knockout mice.

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1-/-) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1-/- mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1-/- mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1-/- and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.

Duodenal cytochrome b (Cybrd 1) and HIF-2α expression during acute hypoxic exposure in mice.

BACKGROUND: Recent evidence suggests that the duodenum can regulate iron absorption independently of hepcidin via the transcription factor Hif-2α acting directly on the transcription of the proteins involved in the iron transport. The current study investigates the temporal relationship between Dcytb and Hif-2α during early hypoxic stimulus in the enterocyte in vivo. METHODS: Duodenal Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. RESULTS: Both Dcytb and Hif-2α protein expression were increased during the first hours of hypoxic duration. A change in hepcidin expression however, was significant only at 72 h hypoxia. Increased iron absorption reported in early hypoxia could be accounted for in part by the enhancement of Dcytb expression by Hif-2α in the duodenum. CONCLUSION: Modulation of Hif-2α predominates over hepcidin in the regulation of intestinal iron absorption during short hypoxic duration. The intestine exerts regulatory mechanisms in the dietary absorption of iron into systemic circulation.

Characterization of the transition-metal-binding properties of hepcidin.

Accumulating evidence suggests that hepcidin, a 25-residue peptide hormone, is the master regulator of iron metabolism. Further evidence suggests that the five N-terminal amino acids are crucial for mediating its biological function. With a histidine residue at position 3, this region also has the potential to bind bivalent metal ions. To characterize this hepcidin-metal interaction in detail, the present study utilizes electrospray MS to measure the binding of a range of metal ions to wild-type and mutant human and murine hepcidins. In addition, the biological effects of these point mutations were tested on Caco-2 and HEK-293T human cell lines and in mice. Our results show that hepcidin-25 can form complexes with copper, nickel and zinc; however, we failed to detect any hepcidin-25 binding to either ferric or ferrous ions. The greatest affinity observed was between hepcidin-25 and copper with a dissociation constant <<1 microM. Substituting the histidine residue at position 3 in human hepcidin-25 and comparably the asparagine residue at position 3 in murine hepcidin-25 with an alanine residue markedly diminished the affinity for copper. The amino acid substitutions also decreased the biological activity of hepcidin-25; namely repression of ferroportin protein levels and hypoferraemia. In summary, the high affinity of hepcidin for copper suggests that hepcidin could bind copper in vivo and this may be of biological relevance.

Recent advances in mammalian haem transport.

Haem is a structural component of numerous cellular proteins and contributes greatly to iron metabolic processes in mammals. Haem-carrier protein 1 (HCP1) has recently been cloned and characterized as a putative transporter in the apical region of the duodenum, and is responsible for uptake of haem into the gut cells. Its expression is regulated pre- and post-translationally in hypoxic and iron-deficient mice, respectively. The identification of HCP1 has revealed the long-sought mechanism by which haem--an important source of dietary iron--is absorbed from the diet by the gut. Feline leukaemic virus receptor (FLCVR) and ABC transporter ABCG2, characterized in haematopoietic cells, have also recently been shown to export haem, particularly under stress. FLVCR protects developing erythroid cells from haem toxicity during the early stages of differentiation, and ABCG2 averts protoporphyrin accumulation (particularly under hypoxic conditions). These haem-efflux proteins are expressed in other cells and tissues including the intestine where they might function as apical haem exporters to prevent toxicity in the enterocytes.

Enhanced insulin signaling and its downstream effects in iron-overloaded primary hepatocytes from hepcidin knock-out mice.

BACKGROUND: Increased body iron stores have been implicated in the pathogenesis of diabetes mellitus. However, the molecular mechanisms involved are unclear. The liver plays a central role in homeostasis of iron and glucose in the body. Mice deficient in hepcidin (the central regulator of systemic iron homeostasis) (Hamp1-/- mice) accumulate iron in the liver in vivo. The effects of such iron loading on hepatic insulin signaling and glucose metabolism are not known. METHODS: Hepatocytes isolated from Hamp1-/- mice were studied for markers of insulin signaling (and its downstream effects), glucose production, expression of gluconeogenic and lipogenic enzymes, and markers of AMPK (AMP-activated protein kinase) activation and oxidative stress. These parameters were studied both in the absence and presence of insulin, and also with the use of an iron chelator. RESULTS: Akt in the insulin signaling pathway was found to be activated in the Hamp1-/- hepatocytes to a greater extent than wild-type (WT) cells, both under basal conditions and in response to insulin. Incubation of the Hamp1-/- hepatocytes with an iron chelator attenuated these effects. There was no evidence of oxidative stress or AMPK activation in the Hamp1-/- hepatocytes. Glucose production by these cells was similar to that by WT cells. Gene expression of key gluconeogenic enzymes was decreased in these cells. In addition, they showed evidence of increased lipogenesis. CONCLUSIONS: Hepatocytes from Hamp1-/- mice showed evidence of greater sensitivity to the effects of insulin than WT hepatocytes. This may explain the insulin-sensitive phenotype that has been reported in classical hemochromatosis.

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism.

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b(561) family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax=3.7 corresponding to a highly anisotropic species, and another at gmax=3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax=2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of +80 mV+/-30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.

A novel association between a SNP in CYBRD1 and serum ferritin levels in a cohort study of HFE hereditary haemochromatosis.

There is emerging evidence that there are genetic modifiers of iron indices for HFE gene mutation carriers at risk of hereditary hemochromatosis. A random sample, stratified by HFE genotype, of 863 from a cohort of 31 192 people of northern European descent provided blood samples for genotyping of 476 single nucleotide polymorphisms (SNPs) in 44 genes involved in iron metabolism. Single SNP association testing, using linear regression models adjusted for sex, menopause and HFE genotype, was conducted for four continuously distributed outcomes: serum ferritin (log transformed), transferrin saturation, serum transferrin, and serum iron. The SNP rs884409 in CYBRD1 is a novel modifier specific to HFE C282Y homozygotes. Median unadjusted serum ferritin concentration decreased from 1194 microg/l (N = 27) to 387 microg/l (N = 16) for male C282Y homozygotes and from 357 microg/l (N = 42) to 69 microg/l (N = 12) for females, comparing those with no copies to those with one copy of rs884409. Functional testing of this CYBRD1 promoter polymorphism using a heterologous expression assay resulted in a 30% decrease in basal promoter activity relative to the common genotype (P = 0.004). This putative genetic modifier of iron overload expression accounts for 11% (95% CI 0.4%, 22.6%) of the variance in serum ferritin levels of C282Y homozygotes.

Activated macrophages induce hepcidin expression in HuH7 hepatoma cells.

BACKGROUND: Hepcidin is an iron regulatory peptide produced by the liver in response to inflammation and elevated systemic iron. Recent studies suggest that circulating monocytes and resident liver macrophages--Küpffer cells--may influence both basal and inflammatory expression of hepcidin. DESIGN AND METHODS: We used an in vitro co-culture model to investigate hepatocyte hepcidin regulation in the presence of activated THP1 macrophages. HuH7 hepatoma cells were co-cultured with differentiated THP1 macrophages for 24 h prior to the measurement of HuH7 hepcidin (HAMP) mRNA expression using quantitative polymerase chain reaction, and HAMP promoter activity using a luciferase reporter assay. Luciferase assays were performed using the wild type HAMP promoter, and constructs containing mutations in BMP/SMAD4, STAT3, C/EBP and E-BOX response elements. Neutralizing antibodies against interleukin-6, interleukin-1beta , and the bone morphogenetic protein inhibitor noggin were used to identify the macrophage-derived cytokines involved in the regulation of HAMP expression. RESULTS: Co-culturing HuH7 cells with differentiated THP1 cells induced HAMP promoter activity and endogenous HAMP mRNA expression maximally after 24 h. This induction was fully neutralized in the presence of an interleukin-1beta antibody, and fully attenuated by mutations of the proximal C/EBP or BMP/SMAD4 response elements. CONCLUSIONS: Our data suggest that the interleukin-1beta and bone morphogenetic protein signaling pathways are central to the regulation of HAMP expression by macrophages in this co-culture model.

Haem and folate transport by proton-coupled folate transporter/haem carrier protein 1 (SLC46A1).

Haem carrier protein 1 (HCP1) was originally identified and characterised as a mammalian haem transporter. However, recent evidence has shown that it is also a proton-coupled folate transporter (PCFT) and mutations in the gene cause hereditary folate deficiency in humans. We therefore investigated haem and folate transport characteristics of PCFT/HCP1 both in vivo and in vitro in CD-1 mice and in the presence or absence of a blocking antibody for PCFT/HCP1, and also in cultured cells (which express PCFT/HCP1 endogenously) to elucidate the specificity and selectivity of PCFT/HCP1. The in vivo study showed that the addition of folic acid inhibited 59Fe-labelled haem transport in hypoxic mice but had no effect in normal mice. Using in vitro methods, the results showed increased [3H]folate uptake into everted duodenum from hypoxic mice but uptake was reduced by the addition of haem or PCFT/HCP1 antibodies to the medium. Caco-2 cells transiently transfected with small interfering RNA (siRNA) PCFT/HCP1 duplex oligos resulted in a 69 % reduction in PCFT/HCP1 mRNA when compared with the control siRNA. Both haem and folate uptake were significantly (P < 0.05) reduced in cells transfected with PCFT/HCP1 siRNA; however, the magnitude of reduction with folic acid uptake was greater (48 %) than that of haem (22.5 %). Overall the data support PCFT/HCP1 as a primary folate transporter with a lower affinity for haem. PCFT/HCP1 could therefore play a physiological role in Fe nutrition and the data highlight the potential for the interaction of folate and haem at the level of intestinal absorption.

Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro.

MDCK cells expressing an inducible duodenal cytochrome b-green fluorescent protein (Dcytb-EGFP) fusion construct were used to investigate the function of Dcytb. The Dcytb-EGFP protein was targeted correctly to the plasma membrane, and cells displayed increased ferric and cupric reductase activities, which were greatly reduced in the presence of doxycycline. The data suggests that Dcytb plays a physiological role in both iron and copper uptake, through divalent metal transporter 1 (DMT1) and copper transporter 1, respectively. In support of this hypothesis, we show that 59Fe uptake was significantly enhanced in Dcytb-EGFP expressing MDCK cells which endogenously express DMT1.

The role of Dcytb in iron metabolism: an update.

Dcytb (duodenal cytochrome b) is an iron-regulated ferric reductase highly expressed in duodenal enterocytes. Its location and strong regulation by iron has indicated it plays an important role in iron absorption. Expression of Dcytb in cells (Caco-2 and MDCK) was found to increase both ferric reductase activity and stimulate uptake of (59)Fe. An additional increase in cupric reductase activity was found in MDCK (Madin-Darby canine kidney) cells expressing Dcytb. Expression and purification of Dcytb in insect cells reveals that Dcytb is a di-haem protein and that the haems are reducible by ascorbate, indicating that ascorbate is the likely intracelluar electron donor. Studies underway in Dcytb-knockout mice reveal that Dcytb is the only iron-regulated ferric reductase in the duodenal mucosa and that loss of Dcytb affects iron absorption.

Duodenal cytochrome B expression stimulates iron uptake by human intestinal epithelial cells.

Duodenal cytochrome B (Dcytb) is localized principally in the apical membrane of the enterocyte. It is thought to act as a ferric reductase that furnishes Fe(II), the specific and selective iron species transported by divalent metal transporter 1 (DMT1) in the duodenal enterocytes. Expression of both genes is strongly iron regulated and is thought to be required for transcellular iron trafficking in concert in response to physiological requirements. We tested this hypothesis by expressing Dcytb in Caco-2 cells, a human cell line model often used to mimic intestinal enterocytes. Iron uptake (59Fe) was significantly higher in Dcytb-transfected Caco-2 cells than in cells transfected with empty vector as a control. Fe(III) reductase activity of Dcytb was measured with ferrozine, a strong chelator of Fe(II) species. Cells expressing Dcytb exhibited enhanced ferric reductase activity as well as increased 59Fe uptake compared with cells transfected with empty vector as a control. Ferrozine blocked iron uptake and preincubation of cells with dehydroascorbate (to increase cellular ascorbate levels) stimulated iron uptake. Cotransfection of Dcytb and DMT1 resulted in an additive increase in iron uptake by the cells. The results confirm Dcytb can act as a ferric reductase that stimulates iron uptake in Caco-2 cells.