Iron metabolism overview: proteins, pathways and general pathology Paul J. Schmidt, PhD Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA [email protected] Introduction Although essential for most life on Earth, iron is highly toxic when present in excess. As a result, iron uptake, distribution, and storage must be closely regulated. Dysregulated iron metabolism may lead to tissue damage and is the cause of a number of human pathological disorders. The average adult contains approximately 4 grams of iron. Almost two-thirds of this total amount is sequestered within hemoglobin of red blood cells (RBCs). It is estimated that almost 200 billion RBC’s need to be replaced each day and nearly 25mg of iron is required to support this erythropoiesis. Reticuloendothelial macrophages phagocytose senescent RBC’s, harvesting the heme and returning 20-25mg of iron into circulation each day. A small amount of iron enters the body daily from the diet (1-2mg) and helps to compensate for the loss of iron through bleeding or epithelial cell sloughing. There is no known means to regulate organismal iron excretion; therefore, iron uptake is closely modulated to maintain whole body iron balance. Insufficient iron leads to restricted erythropoiesis and anemia while elevated iron uptake causes increased tissue loading and damage. General iron homeostasis, and the pathophysiology of iron-related human diseases,1-5 has been reviewed. Furthermore, a large number of animal models have been generated to interrogate iron transport and regulation. 6 Cellular iron storage One method for preventing iron-mediated cellular damage is to assure that the metal is securely sequestered within the cell. Ferritin was the first iron protein characterized and crystallized7 and all mammalian cells are known to produce the protein. This iron storage protein is composed of light and heavy (L-ferritin and H-ferritin) polypeptide chains. The folded protein forms a cage-like structure that can hold an estimated 4500 iron atoms. Ferritin acts as an iron reservoir, isolating excess iron in a safe storage site, and releasing the metal when needed for cellular functions.8 Intracellular Iron Regulatory Proteins In order to maintain appropriate cellular iron regulation, cells sense intracellular iron levels and regulate the expression of iron binding and transport proteins. Iron regulatory proteins (IRP’s) bind to untranslated mRNA sequences termed iron regulatory elements (IRE’s) and modulate transcription of iron transport or storage genes.9 The function of IRP’s is iron regulated. Under iron replete conditions IRP1 contains a 4Fe-4S cluster and is unable to bind IRE’s, while IRP2 undergoes proteosomal degradation.10,11 IRE’s found in the 5’ end of mRNA encoding such proteins as ferritin and ferroportin (FPN1) are bound by IRP’s under conditions of iron scarcity. This binding prevents translation of the mRNA’s by blocking ribosomal assembly. Conversely, the binding of IRP’s to five tandem IRE’s in the 3’ untranslated regions of transferrin receptor 1 (TFR1) or divalent metal transporter (DMT1) greatly stabilizes the mRNA and allows for elevated protein expression and increased iron transport. Dietary Uptake and Distribution of Systemic Iron The uptake of iron from the diet is strictly regulated to assure sufficient metal for RBC production and prevent elevated tissue loading. Non-heme dietary iron enters the body through enterocytes located in the first section of the large intestine. Duodenal enterocytes reduce iron to the ferrous state at the apical membrane and then transport it into the cell through divalent metal transporter 1 (DMT1).12-14 The basolateral transporter ferroportin15-17 transfers iron out of the enterocyte where it is oxidized to the ferric state and binds transferrin (TF) in the plasma. Holo-transferrin is the primary ligand for TFR1 and endocytosis of the complex is the major entryway for iron into most cells. The STEAP family of ferrireductases18 is thought to be required for appropriate intracellular mobilization of iron taken up through the transferrin cycle. Hepcidin is the Master Regulator of Iron Metabolism Hepcidin is an iron-regulated, liver-expressed protein with antimicrobial properties.19-21 The serum concentration of hepcidin is also modulated by inflammation through Interleukin-6 (IL-6) activation of the STAT3 signaling pathway.22,23 Importantly, hepcidin binds to ferroportin, the only known iron exporter. This interaction leads to internalization and degradation of the transporter in duodenal enterocytes and reticulodendothelial macrophages.24 Hepcidin expression is transcriptionally regulated by iron. As organismal iron increases, serum hepcidin becomes elevated and decreases the amount of ferroportin on cell surfaces. This leads to the sequestration of iron in macrophages and diminished dietary iron uptake by duodenal enterocytes. The converse it also true. As iron levels fall, hepcidin expression decreases and more iron is released from the recycling compartment and enters the body from the diet. Finch first postulated that the stores regulator of systemic iron metabolism exists and is essential for the maintenance of appropriate iron balance.25 If the stores regulator fails due to disruptions in the iron regulatory apparatus hepcidin is inappropriately modulated, leading to elevated iron deposition and resulting in hereditary hemochromatosis (HH). It is also known that hepcidin expression is inhibited by anemia, hypoxia26 and in situations of ineffective erythropoiesis. Certain anemias, such as -thalassemia intermedia, are characterized by ineffective erythropoiesis. In this disease hepcidin production is suppressed even in the presence of elevated iron loading.27 Based on this and other observations it appears that the postulated erythroid regulator25 is a more powerful hepcidin modulator than the stores regulator. The erythroid regulator is hypothesized to relay a signal from the site of erythropoiesis (bone marrow) to the liver and diminish hepcidin expression. Erythropoiesis itself downregulates hepcidin expression and bone marrow derived factors are required for this process.28,29 Growth differentiation factor 15 (GDF-15)30 has been postulated to be the erythroid regulator; however, recent work suggests that GDF-15 may not be the mediator.31 Most recently, erythroferrone (ERFE), a hormone mediating hepcidin suppression during early stages of stress erythropoiesis, was discovered.32 Characterization of the erythroid regulator, and its role in hepcidin regulation, is ongoing. Regulators Of Hepcidin Expression Hemojuvelin (HJV) plays a central role in the transcriptional regulation of hepcidin. Loss of functional HJV leads to diminished hepcidin expression, elevated iron uptake and disease.33-35 HJV is a member of the repulsive guidance molecule family (RGM) and a bone morphogenetic protein (BMP) coreceptor.36 TMPRSS6, also known as Matriptase-2, is a liver serine protease. Mutations in TMPRSS6 causes elevated hepcidin levels leading to iron deficiency anemia.37-40 Current thought suggests that TMPRSS6 may function to regulate HJV at the post-translational level by cleaving HJV from the cell membrane and generating a soluble form (sHJV) of the protein.41,42 The HFE protein is an atypical major histocompatibility (MHC) class I-like molecule that binds to TFR143. HFE shares a binding site on TFR1 with its primary ligand TF.44,45 Mutations in HFE are the most common cause of hereditary hemochromatosis.46 Furthermore, TFR2 is a homolog of TFR1 expressed predominantly in the liver.47 Loss of functional HFE or TFR2 results in diminished hepcidin expression and excess iron loading. It has been hypothesized that HFE and TFR2 act as sensors for the serum concentration of TF-Fe2. In this model, HFE in the liver is normally sequestered by TFR1 under conditions of low circulating iron; however, HFE can be displaced from TFR1 through an increase in TF saturation.48 It is uncertain how this mechanism regulates hepcidin expression; however, work in cell culture demonstrated an HFE/TFR2 interaction.49,50 Based on experiments with over-expressd proteins in cell culture51,52 it had been hypothesized that HFE, TFR2 and HJV may form a stable complex that functions to regulate hepcidin expression through HJV. Further work is needed to clarify this iron sensing pathway. Conclusion: The understanding of systemic iron regulation, transport and storage has greatly increased over the past 25 years. A significant number of iron transport and regulatory proteins have been characterized and mutations in many of these genes are known to result in dysregulated iron metabolism and disease. 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