Anita C. G. Chua

The Regulation of Cellular Iron Metabolism

While iron is an essential trace element required by nearly all living organisms, deficiencies or excesses can lead to pathological conditions such as iron deficiency anemia or hemochromatosis, respectively. A decade has passed since the discovery of the hemochromatosis gene, HFE, and our understanding of hereditary hemochromatosis (HH) and iron metabolism in health and a variety of diseases has progressed considerably. Although HFE-related hemochromatosis is the most widespread, other forms of HH have subsequently been identified. These forms are not attributed to mutations in the HFE gene but rather to mutations in genes involved in the transport, storage, and regulation of iron. This review is an overview of cellular iron metabolism and regulation, describing the function of key proteins involved in these processes, with particular emphasis on the livers role in iron homeostasis, as it is the main target of iron deposition in pathological iron overload. Current knowledge on their roles in maintaining iron homeostasis and how their dysregulation leads to the pathogenesis of HH are discussed.

Manganese metabolism is impaired in the Belgrade laboratory rat

Abstract Homozygous Belgrade rats have a hypochromic anaemia due to impaired iron transport across the cell membrane of immature erythroid cells. This study aimed at investigating whether there are also abnormalities of Mn metabolism in erythroid and other types of cells. The experiments were performed with homozygous (b/b) and heterozygous (+/b) Belgrade rats and Wistar rats and included measurements of Mn uptake by reticulocytes in vitro, Mn absorption from in situ closed loops of the duodenum, and plasma clearance and uptake by several organs after intravenous injection of radioactive Mn bound to transferrin (Tf ) or mixed with serum. Similar measurements were made with 59Fe-labelled Fe in several of the experiments. Mn uptake by reticulocytes and absorption from the duodenum was impaired in b/b rats compared with +/b or Wistar rats. The plasma clearance of Mn-Tf was much slower than Mn-serum, but both were faster than the clearance of Fe-Tf. Uptake of 54Mn by the kidneys, brain and femurs was less in b/b than Wistar or +/b rats, but uptake by the liver was greater in b/b rats. Similar differences were found for 59Fe uptake by kidneys, brain and femurs but 59Fe uptake by the liver was also impaired in the liver. It is concluded that the genetic abnormality present in b/b rats affects Mn metabolism as well as Fe metabolism and that Mn and Fe share similar transport mechanisms in the cells of erythroid tissue, duodenal mucosa, kidney and blood-brain barrier.

Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat

Managanese (Mn) is an essential trace element at low concentrations, but at higher concentrations is neurotoxic. It has several chemical and biochemical properties similar to iron (Fe), and there is evidence of metabolic interaction between the two metals, particularly at the level of absorption from the intestine. The aim of this investigation was to determine whether Mn and Fe interact during the processes involved in uptake from the plasma by the brain and other organs of the rat. Dams were fed control (70 mg Fe/kg), Fe-deficient (5–10 mg Fe/kg), or Fe-loaded (20 g carbonyl Fe/kg) diets, with or without Mn-loaded drinking water (2 g Mn/L), from day 18–19 of pregnancy, and, after weaning the young rats, were continued on the same dietary regimens. Measurements of brain, liver, and kidney Mn and nonheme Fe levels, and the uptake of54Mn and59Fe from the plasma by these organs and the femurs, were made when the rats were aged 15 and 63 d. Organ nonheme Fe levels were much higher than Mn levels, and in the liver and kidney increased much more with Fe loading than did Mn levels with Mn loading. However, in the brain the increases were greater for Mn. Both Fe depletion and loading led to increased brain Mn concentrations in the 15-d/rats, while Fe loading also had this effect at 63 d. Mn loading did not have significant effects on the nonheme Fe concentrations.54Mn, injected as MnCl2 mixed with serum, was cleared more rapidly from the circulation than was59Fe, injected in the form of diferric transferrin. In the 15-d-rats, the uptake of54Mn by brain, liver, kidneys, and femurs was increased by Fe loading, but this was not seen in the 63-d rats. Mn supplementation led to increased59Fe uptake by the brain, liver, and kidneys of the rats fed the control and Fe-deficient diets, but not in the Fe-loaded rats. It is concluded that Mn and Fe interact during transfer from the plasma to the brain and other organs and that this interaction is synergistic rather than competitive in nature. Hence, excessive intake of Fe plus Mn may accentuate the risk of tissue damage caused by one metal alone, particularly in the brain.

Hepatic Iron Loading in Mice Increases Cholesterol Biosynthesis

Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up‐regulated with increasing hepatic iron (R2 between 0.602 and 0.164), including those of the rate‐limiting enzyme, 3‐hydroxy‐3‐methylglutarate‐coenzyme A reductase (Hmgcr; R2 = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R2 = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R2 = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol‐regulatory element binding factor 2 (Srebf2; R2 = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron. Conclusion: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity. (HEPATOLOGY 2010;)

Disruption of hemochromatosis protein and transferrin receptor 2 causes iron-induced liver injury in mice†

Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause hereditary hemochromatosis (HH) by impeding production of the liver iron‐regulatory hormone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2, either alone or together, on liver iron loading and injury in mouse models of HH. Iron status was determined in Hfe knockout (Hfe−/−), Tfr2 Y245X mutant (Tfr2mut), and double‐mutant (Hfe−/−×Tfr2mut) mice by measuring plasma and liver iron levels. Plasma alanine transaminase (ALT) activity, liver histology, and collagen deposition were evaluated to assess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase (SOD) activity and F2‐isoprostane levels. Gene expression was measured by real‐time polymerase chain reaction. Hfe−/−×Tfr2mut mice had elevated hepatic iron with a periportal distribution and increased plasma iron, transferrin saturation, and non‐transferrin‐bound iron, compared with Hfe−/−, Tfr2mut, and wild‐type (WT) mice. Hamp1 expression was reduced to 40% (Hfe−/− and Tfr2mut) and 1% (Hfe−/−×Tfr2mut) of WT values. Hfe−/− ×Tfr2mut mice had elevated plasma ALT activity and mild hepatic inflammation with scattered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)–positive cells. Increased hepatic hydoxyproline levels as well as Sirius red and Massons Trichrome staining demonstrated advanced portal collagen deposition. Hfe−/− and Tfr2mut mice had less hepatic inflammation and collagen deposition. Liver F2‐isoprostane levels were elevated, and copper/zinc and manganese SOD activities decreased in Hfe−/−×Tfr2mut, Tfr2mut, and Hfe−/− mice, compared with WT mice. Conclusion: Disruption of both Hfe and Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation, inflammation, and portal fibrosis than was observed with the disruption of either gene alone. The Hfe−/−×Tfr2mut mouse model of iron‐induced liver injury reflects the liver injury phenotype observed in human HH. (HEPATOLOGY 2012)

Dietary iron enhances colonic inflammation and IL-6/IL-11-Stat3 signaling promoting colonic tumor development in mice

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.

Clinical Perspectives on Hereditary Hemochromatosis

Hereditary hemochromatosis (HH) comprises a group of inherited disorders of iron metabolism that can result in progressive iron overload, morbidity, and mortality, generally in adulthood. HFE-related HH is the most common type of HH and will form the core of this discussion. The discovery of new proteins and gene mutations has defined other types of HH, termed non-HFE HH. The regulatory protein hepcidin has a central role in iron homeostasis in these disorders. While the liver is the predominant organ of iron deposition and iron-overload-related disease in HFE-related HH, involvement of extrahepatic tissue can also result in morbidity and mortality if the disorder is not diagnosed before organ damage develops. This review traverses the road from HFE genotype to phenotype with a focus on clinical penetrance, modifier factors for disease expression, and current thoughts and controversies on HH diagnosis and screening.

The Role of Hfe in Transferrin-Bound Iron Uptake by Hepatocytes

HFE‐related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin‐bound iron via transferrin receptor (Tfr) 1 and Tfr1‐independent pathways (possibly Tfr2‐mediated). In this study, the role of Hfe in the regulation of hepatic transferrin‐bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non–iron‐loaded and iron‐loaded wild‐type mice were measured after incubation with 50 nM 125I‐Tf‐59Fe (Tfr1 pathway) and 5 μM 125I‐Tf‐59Fe (Tfr1‐independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real‐time polymerase chain reaction and western blotting, respectively. Tfr1‐mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron‐loaded wild‐type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1‐independent pathway was approximately 100‐fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1‐independent pathway. Conclusion: Tfr1‐mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1‐independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin up‐regulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1‐independent pathway. (HEPATOLOGY 2008.)

Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis

BACKGROUND & AIMS Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. METHODS Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. RESULTS Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. CONCLUSIONS A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice.

Liver and serum iron: discrete regulators of hepatic hepcidin expression.

T o prevent pathological excesses or deficiencies, body iron balance must be tightly controlled due to the lack of a highly evolved mechanism for iron excretion. This is achieved through the liver peptide hepcidin, which efficiently regulates the processes of duodenal iron absorption, macrophage iron release and tissue iron storage, primarily in the liver. Hepcidin is released into the circulation and targets ferroportin, the iron exporter expressed on the surface of duodenal enterocytes, macrophages, and hepatocytes. The binding of hepcidin to ferroportin induces its internalization and degradation, thereby restricting iron entry from the absorptive enterocytes as well as iron release from macrophages and liver iron stores. Hence, appropriate hepcidin expression is paramount for accurate regulation of iron distribution. Indeed, impaired regulation of hepcidin synthesis caused by mutations in key upstream genes in hepcidin regulation—the classical hemochromatosis gene (HFE), transferrin receptor 2 (TFR2), hemojuvelin (HJV), or the hepcidin gene itself (HAMP)—underlies the pathogenesis of the iron overload disorder hereditary hemochromatosis (HH). HJV is a member of the repulsive guidance molecule family and a coreceptor for bone morphogenetic proteins (BMPs), implicating a role for BMP signal transduction in the transcriptional regulation of hepcidin in the liver. The signaling pathway is initiated when BMP binds to its receptors, a complex of BMP receptor (BMPR) types I and II, inducing the phosphorylation of BMPR-I by BMPR-II. This, in turn, activates phosphorylation of the intracellular small mothers against decapentaplegic homologue (SMAD) proteins SMAD1, SMAD5, and SMAD8, which then bind SMAD4, and the complex is translocated to the nucleus, promoting transcription of hepcidin. HJV has been shown to interact directly with BMP6, and their interaction facilitates activation of the BMPR complex and enhances BMP-SMAD signaling to modulate hepcidin expression. Although several BMPs including BMP2, 4, 5, 6, 7, and 9 can stimulate hepcidin expression, BMP6 is physiologically the most relevant. BMP6 is regulated by liver iron levels, increasing with iron loading and decreasing with iron depletion, inducing an up-regulation and down-regulation of Smad1/5/8 phosphorylation and HAMP expression. Studies in Bmp6 null mice have demonstrated that the absence of Bmp6 induces severe iron overload and hepcidin deficiency, highlighting the noncompensatory roles of other functional Bmps. The lack of Bmp6 resulted in inhibition of Smad1/5/8 phosphorylation and their translocation to the nucleus. In contrast, administration of exogenous Bmp6 to mice increased hepatic Hamp expression and reduced both serum iron and transferrin saturation (TS). Liverspecific Smad4 null mice also developed iron overload and impaired Bmp signaling, suppressing hepcidin production. Taken together, these observations strongly support BMP6 as the key endogenous regulator of hepcidin synthesis and iron metabolism in vivo. Recently, it was shown that inhibitory SMAD7 tempers HAMP expression by blocking the interaction of SMAD1/5/8 with SMAD4. TFR2 and HFE are thought to act as iron-sensing molecules to receive signals from circulating holotransferrin to modulate hepatic HAMP expression. TFR2 is a strong candidate as a sensor of serum TS, because it binds holotransferrin and undergoes posttranslational stabilization. As TS increases, HFE dissociates from TFR1 and binds to TFR2 to possibly convey the necessary signal downstream to stimulate hepcidin synthesis. Some studies support the premise that TFR2 and HFE interact with the BMP6–SMAD pathway, because this signaling pathway is impaired in Tfr2 and/or Hfe null mice as well as in subjects with HFE-associated HH, whereas others report no interaction. TFR2 and HFE may also signal independently of each other, because disruption of both Tfr2 and Hfe in mice causes a more severe iron overload phenotype. TFR2 and HFE, however, are likely Abbreviations:: BMP, bone morphogenetic protein; BMPR, BMP receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; HAMP, hepcidin gene; HFE, hemochromatosis gene; HH, hereditary hemochromatosis; HJV, hemojuvelin; LIC, liver iron concentration; MAPK, mitogen-activated protein kinase; SMAD, small mothers against decapentaplegic homologue; TFR2, transferrin receptor 2; TS, transferrin saturation. Address reprint requests to: John K. Olynyk, BMEDSC, MBBS, FRACP, MD, Director of Gastroenterology, Fremantle Hospital, Alma Street, Fremantle 6160, Western Australia, Australia. E-mail: john.olynyk@health.wa.gov.au. CopyrightVC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24449 Potential conflict of interest: Nothing to report.