Regulation of iron balance • Body iron content – 3-4g • Hb, iron containing proteins, bound to Tf, storage (ferritin, haemosiderin). • Iron homeostasis is regulated strictly at level of intestinal absorption.
Intestinal iron absorption • Haem diet – very readily absorbed via haem carrier protein 1 (apical bruish border membrane of duodenal enterocytes) i.e. higher bioavailability. • Remainder of dietary iron poorly absorbed (10%). • Ascorbic acid enhances absorption of non-animal sources of iron; tannates inhibit absorption. • Fe2+ better absorbed cf. Fe3+.
Intestinal iron absorption • Fe3+ freed from food binding sites in stomach, binds to mucin, travels to duodenum and small bowel. • Haem iron - carrier protein (endocytosis). • Fe3+ - attachment to an integrin. • Fe2+ - intestinal transporter DMT1.
Intestinal iron absorption • Iron then enters cytosol, binds to cytosolic low molecular weight iron carriers and proteins e.g. Mobilferrin (shuttles iron with help of ATP) to basolateral membrane • Export from basolateral membrane via duodenal iron exporter.
Iron transport • Upon release into circulation, re-oxidised to Fe3+, loaded onto transferrin. • Site of influence of HFE gene product, +/- caeruloplasmin (known ferroxidase).
Iron regulation • Iron absorption regulated by many stimuli – • Iron stores. • Degree of erythropoiesis (increased with increased erythropoiesis, reticulocytosis). • Ineffective erythropoiesis. • Mobilferrin – mechanism of loss in iron replete state.
Role of specific proteins • Transferrin and TfR. • Ferritin. • Iron responsive element-binding protein (IRE-BP) aka iron regulatory protein/factor (IRP/IRF). • HFE. • Divalent metal transporter (DMT1, Nramp2, DCT1,Slc11a) – duodenal iron transporter. • Ferroportin and hephaestin, iron export proteins. • Hepcidin.
Transferrin (Tf) • Encoded on long arm of chromosome 3. • Half life 8 days. • Hepatic synthesis. • Complete lack incompatible with life (hypotransferrinaemia).
Transferrin receptor (TfR) • Also on long arm of chromosome 3. homodimeric transmembrane protein. • Found in most cells. Most dense on erythroid precursors, hepatocytes, placental cells. • Restricted expression: both TfR1 and TfR2 present at high levels in hepatocytes, epithelial cells of small intestine including duodenal crypt cells.
Transferrin receptor (TfR) • Each TfR binds 2 diferric Tf molecules. Uptake by clustering on clathrin coated pits, then endocytosed. • Iron off-loaded in acidified vacuoles, apotransferrin-TfR complex recycled to cell surface, apo-Tf then released back into circulation.
Ferritin • Cellular storage protein for iron. • L and H chains (chromosome 19, 11). • Synthesis controlled at 2 levels – • DNA transcription via its promotor. • mRNA translation via interactions with iron regulatory proteins. • Acute phase reactant.
Ferritin • Ferritin in erythroid precursors may be of special importance in haem synthesis especially at beginning of Hb accumulation, when Tf-TfR pathway still in sufficient. • When ferritin accumulates, it aggregates, proteolyzed by lysosomal enzymes, , then converted to iron-rich, poorly characterised haemosiderin, which releases iron slowly. • M-ferritin – present in mitochondria. Expression correlated with tissues that have high mitochondrial number, rather than those involved in iron storage.
Iron-regulatory proteins and iron-responsive element binding protein • Sensing iron-regulatory proteins modulate synthesis of TfR, ferritin, DMT1. • IRP1 and IRP2 – cytosolic RNA binding proteins. Bind to iron-responsive elements located in 5’ or 3’ untranslated regions of specific mRNAs encoding ferritin, TfR, DMT1 and (in erythroid cells) eALAS.
IRPs and IRE-BP • Binding of IRPs to IREs at 5’ end of transcrips of e.g. Ferritin, eALAS – decreases rate of synthesis; binding to 3’ end of transcripts e.g. TfR or DMT1, mRNA half life prolonged, increased synthesis. • IRE-IRP complex senses state of iron balance – conformational change.
IRP-IRE-BP • End result – in iron overload, increased ferritin (for adequate storage), decreased TfR (minimise further iron entry into cell), and vice versa in iron deficiency.
HFE protein • Expression in GIT limited to cells in deep crypts in proximity to site of iron absorption. • HFE protein associated with TfR, acts to modulate uptake of Tf-bound iron into crypt cells. • Along with hepcidin, acts as iron sensor. • Hereditary haemochromatosis with HFE gene mutation - inability to bind beta 2-microglobulin, impaired cellular trafficking, reduced incorporation into the cell membrane, reduced association with TfR1.
Duodenal iron transporter • Divalent metal transporter protein – iron transporter (also Pb, Zn, Cu). • Widely expressed, esp. in proximal duodenum. • Isoform containing iron responsive element (Nramp2 isoform I) specifically upregulated in iron deficiency, greatest expression at brush border of apical pole of enterocytes in apical 2/3 of villi.
Duodenal iron transporter • Increased body iron stores – enhanced uptake of iron from circulation into crypt cells. • Increasing intracellular iron into crypt cells, differentiating enterocytes migrating up to villus tip downregulate iron transporter DMT1, reducing absorption of dietary iron from gut. • Inverse relationship between ferritin levels in serum, and DMT1 levels in duodenal cells.
Iron exporters • Transporting iron from basolateral membrane of enterocytes to circulation; from macrophage (from effete RBCs) into circulation for formation of new Hb. • Ferroportin. • Hephaestin.
Ferroportin • Ferroportin-1 in basal portion of placental syncytiotrophoblasts, basolateral surface of duodenal enterocytes, macrophages, hepatocytes. • Upregulated by amount of available iron, downregulated through interaction with hepcidin.
Hephaestin • Mutation in mice with sex-linked anaemia – enterocytes are iron loaded, but efflux through basolateral membrane inhibited. • Homology to caeruloplasmin. • Link between iron deficiency and copper deficiency – administration of copper facilitates egress of iron from tissue(s) into circulation.
Stimulator of iron transport (SFT) • SFT-mediated transport has properties defined for Tf-independent iron uptake, transporting iron across lipid bilayer. Process dependent on Cu. • Has ferrireductase activity. • Cytosolic localisation in recycling endosomes, stimulates Tf bound iron assimilation.
Hepcidin • 25 aa peptide hormone. • Chromosome 19. • Synthesized by hepatocyes. Intrinsic antimicrobial activity.
Hepcidin • Binds ferroportin, complex internalised and degraded. • Resultant decrease in efflux of iron from cells to plasma
Regulation of hepcidin • Iron – stimulated with increased iron levels • Inflammation, infection (and endotoxin) • Hypoxia - downregulated • Erythropoiesis – downregulated in anaemia, oxidative stress, ineffective erythropoiesis.
Hepcidin regulation by bone morphogenic protein pathway • BMP - members of TGF-b superfamily which regulate cell proliferation, differentiation, apoptosis. • Targets BMP receptors type I and II, resulting in phosphorylation of cytoplasmic R-Smads. • R-Smads associate with Smad4, translocate to nucleus, activates transcription of target genes (in this case hepcidin).
Bone morphogenic protein pathway • BMP2, 4, 5, 6, 7, 9 increase hepcidin expression in hepatic cells. • Individual members of BMP family interact with different combinations of type I and II receptors. • BMP’s effect on cellular response also modulated by BMP coreceptors. • Hemojuvelin (HJV) - iron-specific, stimulates BMP2/4 pathway.
Bone morphogenic protein pathway - hemojuvelin • Member of family of repulsive guidance molecules (RGMs) - coreceptors of BMP receptors. • Chromosome 1. • Disruptive mutations cause juvenile haemochromatosis. • 2 forms - • GPI linked membrane form - stimulates BMP signalling and hepcidin expression. • Soluble HJV (sHJV) - antagonist of BMP signalling.
Hepcidin regulation by iron • Production stimulated by increased plasma iron and tissue stores. • Negative feedback - hepcidin decreases release of iron into plasma (from macrophages and enterocytes). • Fe-Tf increases hepcidin mRNA production (dose dependent relationship).
Hepcidin regulation by iron - iron sensors • HFE interacts with TfR1, but dissociates when Fe-Tf binds to TfR1. • Amount of free HFE proportional to Tf-Fe. • TfR2 – Tf-Fe stabilises TfR2 protein in dose dependent fashion. • Fe-Tf binding increases fraction of TfR2 localizing to recycling endosomes, decreases fraction of TfR2 localizing to late endosomes where it is targeted for degradation. • TfR2 competes with TfR1 for binding to HFE. • HFE-TfR2 may regulate hepcidin expression by promoting HJV/BMP signalling, impacting upon hepcidin expression.
Hepcidin regulation by erythropoietic activity • Hepcidin decreased in iron-deficiency anaemia, hereditary anaemias with ineffective erythropoiesis, and mouse models of anaemia from bleeding and haemolysis. Response not seen when erythropoiesis suppressed. • Allows greater availability of iron for erythropoiesis. • Degree of anaemia by itself doesn’t seem as important. • Nature of erythropoietic regulator of hepcidin is unknown – proteins secreted by developing erythrocytes?
Hepcidin regulation by erythropoietic activity • Mechanism particularly important in iron-loading anaemias. • Urinary hepcidin very low in untransfused patients with thalassaemia intermedia, despite high serum and tissue iron levels. • Very high erythropoietic activity overrides hepcidin regulation by iron. • Severe hepcidin suppression leads to increased iron absorption and development of lethal iron overload.
Hepcidin regulation by erythropoietic activity - GDF15 • Member of TGF-b superfamily, mediates hepcidin suppression in thalassaemia. • Secreted during erythroblast maturation. • Suppresses hepcidin mRNA production in primary human hepatocytes. • Uncertain whether GDF15 plays role in pathogenesis other than that of ineffective erythropoiesis. • Levels much lower in sera of sickle cell anaemia, MDS.
Hepcidin regulation by hypoxia • Physiological relevance uncertain. • Hypoxia-inducing factor (HIF) is the main mediator of oxygen-regulated gene expression. • VHL deficiency results in VHL protein deficiency, hence stabilisation of HIF. Resultant decrease in hepcidin levels.
Hepcidin regulation by inflammation • IL-6 a prominent inducer of hepcidin, through STAT-3 dependent transcriptional mechanism. • Other cytokines may also induce hepcidin independent of IL-6. • Macrophage also express hepcidin in response to micobial stimulation. • Hepcidin may function in autocrine manner to degrade macrophage ferroportin, causing local retention of iron in macrophages. • Inflammatory stimuli acting through TNFa suppresses HJV mRNA, thus perhaps preventing iron-regulatory pathway from suppressing hepcidin during hypoferraemia of inflammation.
Iron refractory iron deficiency anaemia (IRIDA) • IDA unresponsive to oral iron supplementation, partially responsive to parenteral iron administration. • Likely autosomal recessive. • ?22q12-13 – encodes type II transmembrane serine protease (matriptase-2), primarily expressed in liver. • Defect in iron uptake.
IRIDA • Elevated urinary hepcidin cf. normal iron deficiency. • ?reason for failure to absorb iron despite iron deficiency. • Still unclear how mutations lead to ainappropriately elevated hepcidin. • Negative regulator of hepcidin transcription in mouse models.