Andrew T. McKie

Identification of an Intestinal Heme Transporter

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption

Background: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. Aims: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. Methods: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive 59Fe. Results: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. Conclusions: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

Modulation of iron transport proteins in human colorectal carcinogenesis

Background and aims: Total body iron and high dietary iron intake are risk factors for colorectal cancer. To date there is no comprehensive characterisation of iron transport proteins in progression to colorectal carcinoma. In this study, we examined expression of iron import (duodenal cytochrome b (DCYTB), divalent metal transporter 1 (DMT1), and transferrin receptor 1 (TfR1)) and export (hephaestin (HEPH) and ferroportin (FPN)) proteins in colorectal carcinoma. Methods: Perl’s staining was used to examine colonocyte iron content. Real time polymerase chain reaction (PCR) and western blotting were used to examine mRNA and protein levels of the molecules of interest in 11 human colorectal cancers. Semiquantitative immunohistochemistry was used to verify protein levels and information on cellular localisation. The effect of iron loading on E-cadherin expression in SW480 and Caco-2 cell lines was examined by promoter assays, real time PCR and western blotting. Results: Perl’s staining showed increased iron in colorectal cancers, and there was a corresponding overexpression of components of the intracellular iron import machinery (DCYTB, DMT1, and TfR1). The iron exporter FPN was also overexpressed, but its intracellular location, combined with reduced HEPH levels, suggests reduced iron efflux in the majority of colorectal cancers examined. Loss of HEPH and FPN expression was associated with more advanced disease. Iron loading Caco-2 and SW480 cells caused cellular proliferation and E-cadherin repression. Conclusions: Progression to colorectal cancer is associated with increased expression in iron import proteins and a block in iron export due to decreased expression and aberrant localisation of HEPH and FPN, respectively. This results in increased intracellular iron which may induce proliferation and repress cell adhesion.

Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

Introduction: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. Methods: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. Results: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. Conclusion: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.

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 SLC40 basolateral iron transporter family (IREG1/ferroportin/MTP1)

The iron regulated-transporter-1 (Ireg1, also known as ferroportin or metal transporter protein-1, MTP1) appears to be the sole member of the SLC40 transporter family. It functions as a universal efflux pathway for iron in a number of cell types. The protein is most highly expressed in mature enterocytes of the duodenum, in syncytiotrophoblasts, which separate foetal and maternal circulations in the placenta, and in macrophages responsible for recycling iron from breakdown of aged red blood cells.

Increased hepcidin expression and hypoferraemia associated with an acute phase response are not affected by inactivation of HFE

The effect of HFE inactivation on iron homeostasis during an acute phase response was investigated in mice. HFE knockout, β2‐microglobulin knockout and C57BL/6J mice were injected with Freunds Complete Adjuvant to induce an acute phase response and hepatic hepcidin expression and serum transferrin saturation was determined 16 h later. Hepcidin mRNA increased in all strains in response to an acute phase stimulus when compared with untreated control animals. Hypoferraemia also occurred in all strains, indicating that both the upregulation of hepcidin and the decrease in transferrin saturation associated with an acute phase response is not dependent on HFE function.

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.

Mechanisms of Haem and Non-Haem Iron Absorption: Lessons from Inherited Disorders of Iron Metabolism

Our current state of knowledge of the mechanism and regulation of intestinal iron absorption has been critically dependent on the analysis of inherited disorders of iron homeostasis in both humans and other animal species. Mutations in DMT1 and Ireg1 have revealed that these molecules are major mediators of iron transport across the brush border and basolateral membranes of the enterocyte, respectively. Similarly, the iron oxidase hephaestin has been shown to play an important role in basolateral iron efflux. The analysis of a range of human iron loading disorders has provided very strong evidence that the products of the HFE, TfR2, hepcidin and hemojuvelin genes comprise integral components of the machinery that regulates iron absorption and iron traffic around the body. Engineered mouse strains have already proved very effective in helping to dissect pathways of iron homeostasis, and in the future they will continue to provide important insights into the absorption of both inorganic and haem iron by the gut.

Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric reductases

Iron has a variety of functions in cellular organisms ranging from electron transport and DNA synthesis to adenosine triphosphate (ATP) and neurotransmitter synthesis. Failure to regulate the homeostasis of iron can lead to cognition and demyelination disorders when iron levels are deficient, and to neurodegenerative disorders when iron is in excess. In this study we show that three members of the b561 family of predicted ferric reductases, namely mouse cytochrome b561 and mouse and fly stromal cell-derived receptor 2 (SDR2), have ferric reductase activity. Given that a fourth member, duodenal cytochrome b (Dcytb), has previously been shown to be a ferric reductase, it is likely that all remaining members of this family also exhibit this activity. Furthermore, we show that the rat sdr2 message is predominantly expressed in the liver and kidney, with low expression in the duodenum. In hypotransferrinaemic (hpx) mice, sdr2 expression in the liver and kidney is reduced, suggesting that it may be regulated by iron. Moreover, we demonstrate the presence of mouse sdr2 in the choroid plexus and in the ependymal cells lining the four ventricles, through in situ hybridization analysis.