Marianne Wessling-Resnick

Chemistry and biology of eukaryotic iron metabolism

With rare exceptions, virtually all studied organisms from Archaea to man are dependent on iron for survival. Despite the ubiquitous distribution and abundance of iron in the biosphere, iron-dependent life must contend with the paradoxical hazards of iron deficiency and iron overload, each with its serious or fatal consequences. Homeostatic mechanisms regulating the absorption, transport, storage and mobilization of cellular iron are therefore of critical importance in iron metabolism, and a rich biology and chemistry underlie all of these mechanisms. A coherent understanding of that biology and chemistry is now rapidly emerging. In this review we will emphasize discoveries of the past decade, which have brought a revolution to the understanding of the molecular events in iron metabolism. Of central importance has been the discovery of new proteins carrying out functions previously suspected but not understood or, more interestingly, unsuspected and surprising. Parallel discoveries have delineated regulatory mechanisms controlling the expression of proteins long known--the transferrin receptor and ferritin--as well as proteins new to the scene of iron metabolism and its homeostatic control. These proteins include the iron regulatory proteins (IRPs 1 and 2), a variety of ferrireductases in yeast an mammalian cells, membrane transporters (DMT1 and ferroportin 1), a multicopper ferroxidase involved in iron export from cells (hephaestin), and regulators of mitochondrial iron balance (frataxin and MFT). Experimental models, making use of organisms from yeast through the zebrafish to rodents have asserted their power in elucidating normal iron metabolism, as well as its genetic disorders and their underlying molecular defects. Iron absorption, previously poorly understood, is now a fruitful subject for research and well on its way to detailed elucidation. The long-sought hemochromatosis gene has been found, and active research is underway to determine how its aberrant functioning results in disease that is easily controlled but lethal when untreated. A surprising connection between iron metabolism and Friedreichs ataxia has been uncovered. It is no exaggeration to say that the new understanding of iron metabolism in health and disease has been explosive, and that what is past is likely to be prologue to what is ahead.

Iron metabolism in the reticuloendothelial system.

Comprised mainly of monocytes and tissue macrophages, the reticuloendothelial system (RES) plays two major roles in iron metabolism: it recycles iron from senescent red blood cells and it serves as a large storage depot for excess iron. Although iron recycling by the RES represents the largest pathway of iron efflux in the body, the precise mechanisms involved have remained elusive. However, studies characterizing the function and regulation of Nramp1, DMT1, HFE, FPN1, CD163, and hepcidin are rapidly expanding our knowledge of the molecular aspects of RE iron handling. This review summarizes fundamental physiological and biochemical aspects of iron metabolism in the RES and focuses on how recent studies have advanced our understanding of these areas. Also discussed are novel insights into the molecular mechanisms contributing to the abnormal RE iron metabolism characteristic of hereditary hemochromatosis and the anemia of chronic disease.

Iron homeostasis and the inflammatory response.

Iron and its homeostasis are intimately tied to the inflammatory response. The adaptation to iron deficiency, which confers resistance to infection and improves the inflammatory condition, underlies what is probably the most obvious link: the anemia of inflammation or chronic disease. A large number of stimulatory inputs must be integrated to tightly control iron homeostasis during the inflammatory response. In order to understand the pathways of iron trafficking and how they are regulated, this article presents a brief overview of iron homeostasis. A major focus is on the regulation of the peptide hormone hepcidin during the inflammatory response and how its function contributes to the process of iron withdrawal. The review also summarizes new and emerging information about other iron metabolic regulators and effectors that contribute to the inflammatory response. Potential benefits of treatment to ameliorate the hypoferremic condition promoted by inflammation are also considered.

Iron metabolism and the innate immune response to infection.

Host antimicrobial mechanisms reduce iron availability to pathogens. Iron proteins influencing the innate immune response include hepcidin, lactoferrin, siderocalin, haptoglobin, hemopexin, Nramp1, ferroportin and the transferrin receptor. Numerous global health threats are influenced by iron status and provide examples of our growing understanding of the connections between infection and iron metabolism.

Attenuated Inflammatory Responses in Hemochromatosis Reveal a Role for Iron in the Regulation of Macrophage Cytokine Translation

Disturbances of iron homeostasis are associated with altered susceptibility to infectious disease, but the underlying molecular mechanisms are poorly understood. To study this phenomenon, we examined innate immunity to oral Salmonella infection in Hfe knockout (Hfe−/−) mice, a model of the human inherited disorder of iron metabolism type I hemochromatosis. Salmonella- and LPS-induced inflammatory responses were attenuated in the mutant animals, with less severe enterocolitis observed in vivo and reduced macrophage TNF-α and IL-6 secretion measured in vitro. The macrophage iron exporter ferroportin (FPN) was up-regulated in the Hfe−/− mice, and correspondingly, intramacrophage iron levels were lowered. Consistent with the functional importance of these changes, the abnormal cytokine production of the mutant macrophages could be reproduced in wild-type cells by iron chelation, and in a macrophage cell line by overexpression of FPN. The results of analyzing specific steps in the biosynthesis of TNF-α and IL-6, including intracellular concentrations, posttranslational stability and transcript levels, were consistent with reduced translation of cytokine mRNAs in Hfe−/− macrophages. Polyribosome profile analysis confirmed that elevated macrophage FPN expression and low intracellular iron impaired the translation of specific inflammatory cytokine transcripts. Our results provide molecular insight into immune function in type I hemochromatosis and other disorders of iron homeostasis, and reveal a novel role for iron in the regulation of the inflammatory response.

A role for the small GTPase Rab21 in the early endocytic pathway.

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.

The Iron Efflux Protein Ferroportin Regulates the Intracellular Growth of Salmonella enterica

ABSTRACT We investigated the influence of the macrophage iron exporter ferroportin and its ligand hepcidin on intracellular Salmonella growth. Elevated ferroportin expression inhibited bacterial multiplication; hepcidin-induced ferroportin down-regulation enhanced it. Expression analysis of iron-responsive Salmonella genes indicated ferroportin-mediated iron deprivation. These results demonstrate a role for ferroportin in antimicrobial resistance.

Olfactory uptake of manganese requires DMT1 and is enhanced by anemia

Manganese, an essential nutrient, can also elicit toxicity in the central nervous system (CNS). The route of exposure strongly influences the potential neurotoxicity of manganese‐containing compounds. Recent studies suggest that inhaled manganese can enter the rat brain through the olfactory system, but little is known about the molecular factors involved. Divalent metal transporter‐1 (DMT1) is the major transporter responsible for intestinal iron absorption and its expression is regulated by body iron status. To examine the potential role of this transporter in uptake of inhaled manganese, we studied the Belgrade rat, since these animals display significant defects in both iron and manganese metabolism due to a glycine‐to‐arginine substitution (G185R) in their DMT1 gene product. Absorption of intranasally instilled 54Mn was significantly reduced in Belgrade rats and was enhanced in iron‐deficient rats compared to iron‐sufficient controls. Immunohistochemical experiments revealed that DMT1 was localized to both the lumen microvilli and end feet of the sustentacular cells of the olfactory epithelium. Importantly, we found that DMT1 protein levels were increased in anemic rats. The apparentfunction of DMT1 in olfactory manganese absorption suggests that the neurotoxicity of the metal can be modified by iron status due to the iron‐responsive regulation of the transporter. Thompson, K., Molina, R. M., Donaghey, T., Schwob, J. E., Brain, J. D., Wessling‐Resnick, M. Olfactory uptake of manganese requires DMT1 and is enhanced by anemia. FASEB J. 21, 223–230 (2007)

Hepcidin Regulation of Iron Transport

The discovery of hepcidin as a key regulator of iron homeostasis has advanced our current knowledge of this field. Liver-derived hepcidin peptide is secreted in response to iron and inflammation and interacts with the iron export protein ferroportin. This review summarizes recent advances discussed at the Symposium. A particular focus is on molecular interactions between hepcidin and ferroportin, the regulation of hepcidin expression by iron and inflammation, and emerging methods to measure serum hepcidin in human populations.

Biochemistry of Iron Uptake

Recent information gained from genetic and biochemical studies of iron transport in yeast, coupled with the identification of specific mutations causing iron uptake disorders in mice and man, has provided new clues about the mechanisms involved in iron uptake. This article summarizes these discoveries and discusses their impact on our current understanding of the biochemistry of iron uptake.