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© 2008 American Society for Nutrition J. Nutr. 138:991-995, June 2008

Biochemical, Molecular, and Genetic Mechanisms

Duodenal Cytochrome B Expression Stimulates Iron Uptake by Human Intestinal Epithelial Cells1,2

Gladys O. Latunde-Dada*, Robert J. Simpson and Andrew T. McKie

Nutritional Sciences Division, School of Biomedical and Health Sciences, King's College London, London, SE1 9HN, UK

* To whom correspondence should be addressed. E-mail: yemisi.latunde-dada{at}kcl.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
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.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Iron is essential for biological processes in virtually all organisms. It is an obligatory cofactor in cellular respiration, replication, and electron recycling. Although its availability is not quantitatively limiting, iron predominates in the trivalent [Fe(III)] form in organic chelates in food, in the circulation bound to transferrin, and in the storage form, ferritin (1). The common bioactive species, Fe(II), participates as a component of biomolecules and enzymes involved in a host of metabolic reactions that sustain life. Consequently, Fe(III) solubilization, reduction, and mobilization are a prerequisite for iron utilization in metabolism. This is achieved in different tissues, cells, and organelles by ferric ion-reducing agents and reductases. Reductases are important as adaptable, responsive, and regulatory agents to control levels and toxicity of the highly reactive ferrous species. Several studies have reported ferric reductase activity (similar to the well-characterized yeast ferric reductase 1 family) in mammalian cultured cells (2–4) and animal duodenal tissues (4–7). A group of ferric reductases that shares a weak homology to the yeast reductases (8,9), the 6-transmembrane epithelial antigen of the prostrate family, has been described (10) and 6-transmembrane epithelial antigen of the prostrate 3 is important as the endosomal reductase in erythroid cells (11). Moreover, cytochrome b561 family proteins have also been reported as tissue-specific ascorbate-dependent ferrireductases in chromaffin granules, lysosomes, and erythrocytes (12,13). The acquisition of exogenous iron by mammals, although remotely regulated by hepatocytes (14,15), occurs solely in the duodenum (16). Under normal conditions, the amount of iron absorbed through this route is small relative to internal turnover; however, it is important in preventing whole-body iron accumulation or deficit, as the modulation of iron excretion is limited. Absorption is achieved principally by divalent metal transporter-1 (DMT1)3 (17,18), the main Fe(II) transporter at the apical surface of the enterocyte. Food iron mainly in Fe(III) form is consequently thought to be reduced by duodenal cytochrome B (Dcytb), the apical ferric reductase (19,20) that furnishes DMT1 with ferrous ion for transport into the enterocyte (19). Although dietary ferric iron may be reduced by other dietary reductants, as well as other membrane reductases, Dcytb remains the only known iron-regulated reductase in the duodenum. Dcytb is expressed predominantly in the duodenum and both the mRNA and protein are up-regulated by several stimulators of iron absorption (19,21,22).

Although Dcytb exhibited ferric reductase activity when expressed in Xenopus oocytes and cultured cells, this was not linked with its functional capability (19). This finding is imperative, because a recent study reported a lack of an iron-deficient phenotype (23) in Dcytb null mice, thereby suggesting a functional overlap and redundancy in the ferrireduction system. Although that report did not include experiments to test the effect of Dcytb knockout in all circumstances of enhanced dietary iron absorption (24), the evidence was nevertheless compelling that Dcytb was not required for iron absorption during normal growth of mice fed a standard laboratory diet. This study reports data on the expression and function of Dcytb in Caco-2 cells, an in vitro human intestinal model system, including its effect on iron uptake.


   Materials and Methods
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 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Cell culture. Caco-2 cells were obtained from the American Type Culture Collection. Cells (passage 45) were cultured in DMEM (Sigma) medium supplemented with 10% fetal calf serum (Sigma) and with 100 kU/L of penicillin, 100 mg/L streptomycin, and 1% minimum essential amino acids. Cells were maintained at 37°C in a humidified incubator containing 95% air/5% CO2. Cells were trypsinized, plated in 24-well plates, and grown for 24 h to 60–70% confluency for the experiments.

    Transfection of Caco-2 cells. Human Dcytb was excised from the original vector pMEL18S-FL/Dcytb Gen Bank accession number AK027115 with EcoR1 and Xho1 and subcloned into pcDNA 3.1 myc/his(+) mammalian expression vector (Invitrogen). Rat DMT1-iron response element was also subcloned into pcDNA 3.1 myc/his with Kpn1 and EcoRV. Caco2 cells were grown to 60–70% confluency and were transiently transfected with plasmid DNA using Effectene (a nonliposomal lipid formulation) according to the manufacturer's protocol (Qiagen). Control cells were transfected with empty pcDNA 3.1 vector.

    Ferric reductase and iron uptake assays. Ferric-nitrilotriacetate reduction was measured on Caco-2 cells as described previously (19) measuring ferrozine-chelable Fe(II) at an absorbance of 562 nm. Ferric reductase and uptake assays were conducted on cells after 72 h of transfection with Dcytb and the empty vector control.

Iron uptake was assayed in Hank's Balanced Salt Solution buffer (pH 7.0) and freshly prepared 59Fe(III)-NTA (1:2) in a final concentration of 10 µmol/L iron. Uptake was initiated by the addition of 250 µL of assay buffer to the cells and terminated after 60 min unless otherwise stated. Cells were washed 3 times in ice-cold Versene (0.2 g EDTA/L PBS) to remove nonspecifically bound 59Fe. Cells were collected in 200 µL of 2% sodium deoxycholate in 10 mmol/L Tris and radioactivity measured with a gamma counter (LKB Wallace 1280). Then 20 µL of the cell extract was used for protein concentration determination according to Bio-Rad assay protocol (Bio-Rad Laboratories).

    Western blot analysis. Transiently transfected cells grown for 72 h were harvested in Laemmli buffer and subjected to a 10% SDS-PAGE as described earlier (19). Western blotting was performed on the nitrocellulose onto which the proteins had been transferred. Membrane was incubated for 1 h with Dcytb antisera (Sigma Genosys, Sigma-Aldrich) at a dilution of 1:100 (19). Detection of the primary antibody was performed with the Westernbreeze system (Invitrogen).

    Immunohistochemistry. Protein expressions were localized by immunofluorescence microscopy on cells grown and transfected with Dcytb in Chamber slides. Staining of cells was done 72 h after transfection. Briefly, cells were fixed in formaldehyde, blocked with 1% bovine serum albumin, and stained with both Dcytb polyclonal antibody (1:100 dilution) and fluorescein isothiocyanate-conjugated Ig (DAKO A/S) according to standard immunohistochemical protocols.

    Statistical analysis. All values are expressed as means ± SEM. Statistical differences between means were calculated with Microsoft Excel 6.0 by using the Student's t test correcting for differences in sample variance. When multiple comparisons were necessary, 1-way or 2-way ANOVA was performed, using SPSS 14 with Tukey's post hoc test. Differences were considered significant at P < 0.05.


   Results
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Reductase activity and Dcytb protein expression. Dcytb protein is expressed in Caco-2 cells transiently transfected with Dcytb cDNA (Fig. 1A). Dcytb protein stained green and appeared predominantly intracellular colocalized with the apical membrane even in cells examined 72 h after transfection. Transfection efficiency was low and the pockets of Dcytb-expressing cells were randomly dispersed on the slide (Fig. 1A). The nuclei were stained red with propidium iodide. Expression of Dcytb protein in the transfected cells was confirmed further by Western blot analysis (Fig. 1B). As our earlier studies showed (25), endogenous expression of Dcytb is very low as evident by immunohistochemical and Western blot analyses. These cells mediated ferric iron reduction and enhancement of reduction by Dcytb transfection was 48%, as previously reported (19). Enhanced protein expression was also seen in the Western blot analysis of the cells overexpressing Dcytb (Fig. 1B).


Figure 1
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  		FIGURE 1  Expression of Dcytb in Caco-2 cells. (A) Immunohistochemical staining of Dcytb in cells (magnification x40). Dcytb protein expression was stained green in Caco-2 cells transiently transfected with the plasmid. Cells were stained with both Dcytb polyclonal antibody and fluorescein isothiocyanate-conjugated Ig. The nuclei are stained red with propidium iodide. The endogenous level of Dcytb was virtually nil in cells transfected with empty vector control (B). Western blot analysis of the cells after transfection with Dcytb. The ~30-kDa protein was highly expressed in the transfected cells.

 
    Iron uptake requires Fe(III) reduction and transport of Fe(II). Iron uptake increased progressively with the time course of the assay (Fig. 2A). Maximal uptake occurred during the 1-h incubation period and this time point was used for subsequent assays. Iron uptake was significantly higher in Dcytb-transfected Caco-2 cells than in cells transfected with empty vector as a control. Furthermore, Dcytb antibody significantly inhibited iron uptake in Dcytb-transfected cells (Fig. 2B). Pretreating the Caco-2 cells with 50 µmol/L dehydroascorbic acid enhanced iron uptake in both the washed vector control and Dcytb-transfected cells (Fig. 3). Ferrozine [a specific Fe(II) chelator] inhibited uptake of 59Fe, indicating that Fe(III) reduction precedes iron transport (Fig. 3).


Figure 2
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  		FIGURE 2  Time-course 59Fe uptake (A) and effects of inhibition of iron uptake by Dcytb antibody (B) in Caco-2 cells. Uptake buffer contained 10 µmol/L Fe and incubation times are as indicated. Dcytb antibody (Ab) but not the preimmune (PI) (serum inhibited iron uptake in Caco-2 cells transfected with Dcytb). Values are means ± SEM, n = 4. A: *Different from 0 min, P < 0.05; **different from Caco-2 at that time, P < 0.05; B: means in a column without a common letter differ, P < 0.05.

 

Figure 3
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  		FIGURE 3  Effect of DHA (50 µmol/L) and ferrozine (Fe II chelator) on iron uptake. in Caco-2 cells that were or were not transfected with Dcytb. Values are means ± SEM, n = 4. DHA increased (P < 0.001) and ferrozine decreased (P < 0.001) iron uptake. Dcytb transfection did not interact with DHA treatment but did with ferrozine treatment (P < 0.02). Logarithmic transformation of the data did not change the significance of the main effects but removed the significant interaction between ferrozine treatment and Dcytb transfection. We conclude that treatment with ferrozine had a similar proportional effect on iron uptake in untransfected and Dcytb-transfected cells.

 
    Cotransfection of Dcytb and DMT1 plasmids into Caco-2 cells. Iron uptake was significantly enhanced when Dcytb and DMT1 were independently transfected into Caco-2 cells (Fig. 4). Cotransfection of both Dcytb and DMT1 resulted in an additive effect in the enhancement of 59Fe uptake over the control (Fig. 4). Thus, enhanced ferrireductase activity was either sequentially or concurrently coupled to accelerated iron uptake.


Figure 4
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  		FIGURE 4  Iron uptake in Caco-2 cells transfected with Dcytb, DMT1, or both. Equal amounts of plasmid DNA (0.2 µg) were transiently transfected into cells. Values are means ± SEM, n = 6. Transfection with DMT1 and Dcytb increased iron uptake and there was no interaction between them. We conclude that the effects of DMT1 and Dcytb transfection were additive.

 

   Discussion
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Caco-2 cells have proven to be a useful model for studying the characteristics of intestinal iron uptake and metabolism (26,27) and were therefore employed in the current study to evaluate the role of Dcytb in iron uptake. Heterologous expression of Dcytb in Caco-2 cells confirms its ferric reductase activity as reported previously (19). Moreover, current data show its enhancing effect on iron uptake (Fig. 2). The latter was confirmed by the fact that ferrozine, a strong and membrane-impermeable Fe(II) chelator, competed for available ferrous ion and inhibited uptake (Fig. 3). The data demonstrates further that an apical surface ferric reductase is essential for Fe(III) conversion to Fe(II) before it is taken up by the cells (2). Inhibition of ferrireductase activity has been shown to decrease Caco-2 cell's apical iron uptake (26). Moreover, extracellular ferrireductase activity has been shown to be coupled to transferrin-independent iron transport in K562 cells (28). Enhanced expression of Dcytb possibly resulted in increased available Fe(II) for transport by DMT1 into cells. Although the endogenous expression of Dcytb is low (Fig. 1A,B), there was modest ferric reductase and iron uptake activities by these cells. This might suggest the presence of other surface ferric reductases or extracellular-reducing agents, possibly in the assay medium. Dcytb has been shown in several studies to be the most highly regulated gene of the iron absorption pathway at the mRNA level (21,22,29). Our data show that enhanced expression of Dcytb results in increased iron uptake, possibly by promoting the availability of reduced iron for the transporter. As also demonstrated previously (17,30,31), transfection of Caco-2 cells with DMT1 exerted an enhancing effect on iron uptake at the physiological pH employed in this study. The magnitude of increase in iron uptake in DMT1-expressing cells is low and this might be due to relative differences in the transient transfection efficiency of the plasmid DNAs. Although DMT1 activity is maximal at a slightly acidic pH of 5–6, substantial iron uptake has been shown to occur at pH 7.0 (30), which we chose for our experiments. It is important, however, that cotransfection of Dcytb and DMT1 exhibited an additive effect on iron uptake in the cells (Fig. 4). Although both proteins seem to be coordinately regulated by hepcidin (21) under in vivo conditions, DMT1 might be the rate determinant in the iron uptake process. This kinetic regulation would be cytoprotective and insulate cells from damage associated with unrestricted influx of excess Fe(II) that could potentially produce reactive free radicals. Whereas selective knockout of DMT1 in the intestine resulted in an iron-deficient phenotype (32), that of Dcytb did not have an large effect on iron status in the mice (23). However, under specific dietary or physiological conditions, Dcytb may be required in vivo and thus further investigation is necessary. It is well documented, however, that nonenzymatic reduction of Fe(III) is feasible by luminal reducing agents, e.g. ascorbic acid.

Interestingly, Dcytb has a conserved ascorbic acid-binding motif and is thought to use cytoplasmic ascorbate to generate reducing equivalents for ferric reduction and ascorbate recycling, respectively (33). Preincubation of Caco-2 cells with dehydroascorbic acid (DHA) increased iron uptake, particularly in Dcytb-transfected cells (Fig 3). DHA can be taken up by cells and converted to ascorbate intracellularly where this appears to have generated reducing equivalents for Dcytb reductase activity. In essence, cytosolic ascorbate provides electrons for Dcytb for surface or apical reduction of Fe(III) to Fe(II). Interestingly, mucosal ascorbic acid levels were correlated with Fe(III) reduction rates and adaptive responses to iron absorption in mice (33) and humans (34).

Reduction of iron is indispensable for DMT1, because it discriminates against ferric but selectively transports ferrous ion (23). Dcytb, appears to be the principal iron-regulated ferric reductase, expressed in the duodenum, operating at the apical region of the enterocytes. Both proteins seem to exert some form of cooperativity and synchrony of expression (24) and functional activities in the regulation of iron uptake. Although the situation under in vivo conditions could be complicated by confounding influences of systemic variables and redundancy exerted by homologs, substrates, and carrier plurality, the present in vitro approach has identified a direct response of iron uptake to enhanced Dcytb expression.


   FOOTNOTES
 
1 Supported by UK Biotechnology and Biological Sciences Research Council, Medical Research Council, and Wellcome Trust. Back

2 Author disclosures: G. O. Latunde-Dada, R. J. Simpson, and A. T. McKie, no conflicts of interest. Back

3 Abbreviations used: Dcytb, duodenal cytochrome b; DHA, dehydroascorbic acid; DMT1, divalent metal transporter 1. Back

Manuscript received 5 October 2007. Initial review completed 24 November 2007. Revision accepted 1 April 2008.


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