2.  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.

3.  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+.

4.  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.

5.  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.

6.  Upon release into circulation, re-oxidised to Fe3+, loaded onto transferrin. ◦ Site of influence of HFE gene product, +/- caeruloplasmin (known ferroxidase).

7.  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.

9.  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.

10.  Encoded on long arm of chromosome 3.  Half life 8 days.  Hepatic synthesis.  Complete lack incompatible with life (hypotransferrinaemia).

11.  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.

12.  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.

13.  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.

14.  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.

15.  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.

16.  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.

17.  End result – in iron overload, increased ferritin (for adequate storage), decreased TfR (minimise further iron entry into cell), and vice versa in iron deficiency.

21.  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.

22.  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.

23.  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.

24.  Transporting iron from basolateral membrane of enterocytes to circulation; from macrophage (from effete RBCs) into circulation for formation of new Hb. ◦ Ferroportin. ◦ Hephaestin.

25.  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.

27.  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.

28.  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.

29.  25 aa peptide hormone.  Chromosome 19.  Synthesized by hepatocyes. Intrinsic antimicrobial activity.

30.  Binds ferroportin, complex internalised and degraded.  Resultant decrease in efflux of iron from cells to plasma

31.  Iron – stimulated with increased iron levels  Inflammation, infection (and endotoxin)  Hypoxia - downregulated  Erythropoiesis – downregulated in anaemia, oxidative stress, ineffective erythropoiesis.

32.  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).

33.  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.

34.  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.

35.  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).

36.  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.

38.  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?

39.  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.

40.  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.

41.  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.

42.  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.

44. Iron overloadIron deficiency Hypotransferrinaemia - recessive HFE gene mutation TfR2 gene mutation – recessive Ferroportin mutation – autosomal dominant Hepcidin mutations Hemojuvelin mutations H ferritin mutation - dominant TMPRSS6 mutation - IRIDA

46.  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.

48.  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.