Deepak Darshan

Intestinal ferritin H is required for an accurate control of iron absorption.

To maintain appropriate body iron levels, iron absorption by the proximal duodenum is thought to be controlled by hepcidin, a polypeptide secreted by hepatocytes in response to high serum iron. Hepcidin limits basolateral iron efflux from the duodenal epithelium by binding and downregulating the intestinal iron exporter ferroportin. Here, we found that mice with an intestinal ferritin H gene deletion show increased body iron stores and transferrin saturation. As expected for iron-loaded animals, the ferritin H-deleted mice showed induced liver hepcidin mRNA levels and reduced duodenal expression of DMT1 and DcytB mRNA. In spite of these feedback controls, intestinal ferroportin protein and (59)Fe absorption were increased more than 2-fold in the deleted mice. Our results demonstrate that hepcidin-mediated regulation alone is insufficient to restrict iron absorption and that intestinal ferritin H is also required to limit iron efflux from intestinal cells.

Interacting signals in the control of hepcidin expression

The amount of iron in the plasma is determined by the regulated release of iron from most body cells, but macrophages, intestinal enterocytes and hepatocytes play a particularly important role in this process. This cellular iron efflux is modulated by the liver-derived peptide hepcidin, and this peptide is now regarded as the central regulator of body iron homeostasis. Hepcidin expression is influenced by systemic stimuli such as iron stores, the rate of erythropoiesis, inflammation, hypoxia and oxidative stress. These stimuli control hepcidin levels by acting through hepatocyte cell surface proteins including HFE, transferrin receptor 2, hemojuvelin, TMPRSS6 and the IL-6R. The surface proteins activate various cell signal transduction pathways, including the BMP-SMAD, JAK-STAT and HIF1 pathways, to alter transcription of HAMP, the gene which encodes hepcidin. It is becoming increasingly apparent that various stimuli can signal through multiple pathways to regulate hepcidin expression, and the interplay between positive and negative stimuli is critical in determining the net hepcidin level. The BMP-SMAD pathway appears to be particularly important and disruption of this pathway will abrogate the response of hepcidin to many stimuli.

Regulation of systemic iron homeostasis: how the body responds to changes in iron demand

The iron that is required to meet the metabolic needs of cells and tissues is derived from the plasma. Plasma iron in turn reflects the release of iron from various body cells, principally the macrophages of the reticuloendothelial system, and the absorption of dietary iron by the proximal small intestine. This iron donation is highly regulated and the liver-derived peptide hepcidin has emerged as the key modulator of cellular iron export. Following its synthesis and secretion from the liver, circulating hepcidin reduces iron export into the plasma by binding to the iron efflux protein ferroportin1 on the surface of enterocytes, macrophages and other cell types and causing its internalization. The level of hepatic hepcidin expression is influenced by HFE, transferrin receptor 2 and hemojuvelin, and the signal transduction pathway(s) linking these proteins to hepcidin are only beginning to be revealed. Hemojuvelin has recently been shown to signal through the bone morphogenetic protein pathway, ultimately activating receptor SMAD/SMAD4 complexes to alter hepcidin transcription. Circulating differic transferrin has emerged as a possible upstream regulator of the liver-based hepcidin regulatory pathway. In addition to being regulated by body iron requirements, hepcidin expression can be modulated by pro-inflammatory cytokines such as interleukin-6. The continuing analysis of inherited disorders of iron metabolism combined with biochemical analysis of signal transduction pathways is essential to fully define this important regulatory system.

Metabolic Adaptation to Tissue Iron Overload Confers Tolerance to Malaria

Disease tolerance is a defense strategy that limits the fitness costs of infection irrespectively of pathogen burden. While restricting iron (Fe) availability to pathogens is perceived as a host defense strategy, the resulting tissue Fe overload can be cytotoxic and promote tissue damage to exacerbate disease severity. Examining this interplay during malaria, the disease caused by Plasmodium infection, we find that expression of the Fe sequestering protein ferritin H chain (FtH) in mice, and ferritin in humans, is associated with reduced tissue damage irrespectively of pathogen burden. FtH protection relies on its ferroxidase activity, which prevents labile Fe from sustaining proapoptotic c-Jun N-terminal kinase (JNK) activation. FtH expression is inhibited by JNK activation, promoting tissue Fe overload, tissue damage, and malaria severity. Mimicking FtHs antioxidant effect or inhibiting JNK activation pharmacologically confers therapeutic tolerance to malaria in mice. Thus, FtH provides metabolic adaptation to tissue Fe overload, conferring tolerance to malaria.

Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule-specific FtH-knockout mice (FtH(PT-/-) mice). FtH(PT-/-) mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtH(PT-/-) mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase-associated lipocalin, hemopexin, and transferrin were increased in FtH(PT-/-) mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Molecular basis of iron-loading disorders

Iron-loading disorders (haemochromatosis) represent an important class of human diseases. Primary iron loading results from inherited disturbances in the mechanisms regulating intestinal iron absorption, such that excess iron is taken up from the diet. Body iron load can also be increased by repeated blood transfusions (secondary iron loading), usually as part of the treatment for various haematological disorders. In these syndromes, an element of enhanced iron absorption is also often involved. The central regulator of body iron trafficking is the liver-derived peptide hepcidin. Hepcidin limits iron entry into the plasma from macrophages, intestinal enterocytes and other cells by binding to the sole iron-export protein ferroportin, and facilitating its removal from the plasma membrane. Mutations in hepcidin or its upstream regulators (HFE, TFR2, HFE2 and BMP6) lead to reduced or absent hepcidin expression and a concomitant increase in iron absorption. Mutations in ferroportin that prevent hepcidin binding produce a similar result. Increased ineffective erythropoiesis, which often characterises erythrocyte disorders, also leads to reduced hepcidin expression and increased absorption. Recent advances in our understanding of hepcidin and body iron homeostasis provide the potential for a range of new diagnostic and therapeutic tools for haemochromatosis and related conditions.

Conditional deletion of ferritin H in mice induces loss of iron storage and liver damage

Ferritin plays a central role in iron metabolism by acting both as iron storage and a detoxifying protein. We generated a ferritin H allele with loxP sites and studied the conditional ferritin H deletion in adult mice. Ten days after Mx‐Cre induced deletion, ferritin H messenger RNA (mRNA) was below 5% in the liver, spleen, and bone marrow of deleted mice compared to control littermates. Mice lost their cellular iron stores indicating the requirement of ferritin H in iron deposition. Serum iron and transferrin saturation were slightly increased and correlated with a two‐fold increased liver hepcidin 1 mRNA and a reduced duodenal DcytB mRNA level. Under a normal iron regimen, deleted mice survived for 2 years without visible disadvantage. Mice fed on a high iron diet prior to ferritin H deletion suffered from severe liver damage. Similarly, ferritin H deleted mouse embryonic fibroblasts showed rapid cell death after exposure to iron salt in the medium. This was reversed by wild‐type ferritin H but not by a ferritin H mutant lacking ferroxidase activity. Cell death was preceded by an increase in cytoplasmic free iron, reactive oxygen species, and mitochondrial depolarization. Conclusion: Our results provide evidence that the iron storage function of ferritin plays a major role in preventing iron‐mediated cell and tissue damage. (HEPATOLOGY 2009.)

Stimulated erythropoiesis with secondary iron loading leads to a decrease in hepcidin despite an increase in bone morphogenetic protein 6 expression

The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbbth3/+ mouse, a model of the iron loading anaemia β‐thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbbth3/+ mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.

The multicopper ferroxidase hephaestin enhances intestinal iron absorption in mice

Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.

Severe iron deficiency blunts the response of the iron regulatory gene Hamp and pro-inflammatory cytokines to lipopolysaccharide

Background Expression of the key iron regulatory hormone hepcidin is increased by some stimuli (iron loading, inflammation) but decreased by others (increased erythropoiesis, iron deficiency). We investigated the response of hepcidin to increased erythropoiesis and iron deficiency in the presence of an acute inflammation to assess the relative strengths of these stimuli. Design and Methods Sprague-Dawley rats were maintained on control or iron-deficient diets and treated with lipopolysaccharide to induce inflammation or phenylhydrazine to stimulate erythropoiesis. The levels of Hamp, IL-6 and α2m mRNA were determined by qualitative real-time polymerase chain reaction and those of serum interleukin-6 and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. Cultured RAW264.7 and HuH7 cells were used in associated studies. Results The increase in hepatic hepcidin levels induced by lipopolysaccharide was not affected by phenylhydrazine treatment but was blunted by iron deficiency. Lipopolysaccharide-treated iron-deficient animals also showed lower liver α2m mRNA and reduced serum interleukin-6 and tumor necrosis factor-α, suggesting a more generalized effect of iron deficiency. Similarly, RAW 264.7 cells treated with iron chelators and then stimulated with lipopolysaccharide showed lower IL-6 mRNA than cells treated with lipopolysaccharide alone. Huh7 cells treated with an iron chelator showed a blunted hepcidin response to interleukin-6, suggesting that the response of hepatic parenchymal cells to inflammatory cytokines may also be iron-dependent. Conclusions In any one physiological situation, net hepcidin levels are determined by the relative strengths of competing stimuli. The ability of severe iron deficiency to blunt the response to lipopolysaccharide of both hepcidin and other markers of inflammation suggests that adequate iron levels are necessary for a full acute phase response.