Molecular basis of iron homeostasis
and its translation to the clinic
Tomas Ganz, PhD, MD Systemic iron homeostasis Liver (storage, recycling) Spleen (recycling, storage)
Liver Fe
20-25 mg/day Plasma Erythrocytes Fe-Tf 1-2 mg/day
1 ml of PRBC = 1 mg Fe Bone marrow (usage) Duodenum (absorption) Losses 1-2 mg/day During erythropoietic stimulation Liver (storage, recycling) Spleen (recycling, storage)
Liver Fe
Plasma Erythrocytes Fe-Tf
1 ml of PRBC = 1 mg Fe Bone marrow (usage) Duodenum (absorption) Losses Erythropoietin During infection/inflammation Liver (storage, recycling) Spleen (recycling, storage)
Liver Fe Inflammation
Plasma Erythrocytes Fe-Tf
1 ml of PRBC = 1 mg Fe Bone marrow (usage) Duodenum (absorption) Losses Hepcidin—an iron-regulatory peptide hormone • Made in the liver as 84 aa preprohepcidin • Cleaved to 25 aa bioactive hepcidin by furin • Secreted by hepatocytes • Regulates intestinal iron absorption and the distribution of iron to tissues
DTHFPICIFCCGCCHRSKCGMCCKT
Jordan et al. JBC 2009 Tomas Ganz Hepcidin peptide lowers plasma iron
Synthetic hepcidin 50 µg IP/mouse
40
M) 30
20 * * *
Serum IronSerum ( * 10 * *
0 0 1 2 3 4 5 6 24 48 72 96 Time (hours) Rivera et al. Blood 2005 Ferroportin • 12 transmembrane segment protein • Found in plants, invertebrates, vertebrates • In vertebrates, the receptor for hepcidin • The only cellular iron exporter known (Donovan et al.) • Present in the macrophages, duodenum, hepatocytes, and the placenta (Donovan et al., McKie et al., Abboud et al.)
45 61 115 127 203 206 324 343 393 450 512 518
23 80 96 152 186 228 307 362 374 471 493 537
N C Ferroportin is internalized by hepcidin and degraded in lysosomes
control + 1 µg/ml hepcidin
Nemeth et al. Science 2004 Regulation of intestinal iron absorption Dietary iron uptake Food Dietary iron uptake
ferritin ferritin Duodenal enterocytes Fpn
Fpn X Blood hepcidin Fe Fe Low hepcidin High hepcidin Regulation of erythrocyte iron recycling
Erythrocyte Erythrocyte uptake uptake Macrophages
ferritin ferritin
Fpn
Fpn X Iron release into plasma hepcidin Fe Fe Low hepcidin High hepcidin Hepcidin regulates dietary iron absorption and influx to plasma
Liver Spleen
Liver hepcidin Fe Fpn hepcidin Fpn
Plasma Fe-Tf Erythrocytes Fpn
Bone marrow Duodenum
Tomas Ganz Signals regulating hepcidin Inflammation Liver Spleen
Liver hepcidin Fe Fpn hepcidin Fpn
Plasma Erythrocytes Fe-Tf Fpn Iron signals
Bone marrow Duodenum
Erythropoietic signal Hepcidin excess in anemia of inflammation • Hepcidin-dependent effects – Inflammatory cytokines increase hepcidin and cause Fe trapping in macrophages – Fe restricts hemoglobin synthesis and erythropoiesis is inhibited • Hepcidin-independent effects – Shortened erythrocyte lifespan – Direct suppression of erythropoiesis by cytokines
Iron-restricted anemias
Inflammation Liver Spleen
Plasma hepcidin clearance Fe-Tf RBC
Duodenum Bone Kidneys marrow Ablation of hepcidin is beneficial in anemia of inflammation induced by heat-killed Brucella abortus
18
16 hepcidin KO WT 14 saline
12 Brucella 10
8 Hemoglobin (g/dL)
6 0 7d 14d 21d 28d Time
Kim et al. Blood 2014 Hepcidin deficiency in β-thalassemia
secreted suppressors less hepcidin Fe overload Worse anemia
• Failure of red cell precursors to mature into functional RBCs • Anemia, increased erythropoietin • Expansion of immature RBC precursors • Hepcidin-suppressive signal causes hepcidin deficiency Anemia • Increased Fe absorption, Fe overload Rational design of peptide agonists • Strategy for development: structure-function study of hepcidin and ferroportin
C
N 45 61 115 127 203 206 324 343 393 450 512 518
23 80 96 152 186 228 307 362 374 471 493 537
N C
Hepcidin Ferroportin 25 aa peptide hormone 571 aa protein 4 disulfide bonds 12 transmembrane segments
Preza at al. JCI 2011, Ramos et al. Blood 2012 Minihepcidin PR73
Ida-Thr-His-Dpa-bhPro-Arg-Cys-Arg-bhPhe-Ahx- Ida(NHPal)-CONH2 iminodiacetic acid, L-threonine, L-histidine, L-3,3-diphenylalanine, L-β-homoproline, L- arginine, L-cysteine, L-arginine, L-β-homophenylalanine, 6-aminohexanoic acid, iminodiacetic acid palmitylamide (PEG-free) Mouse model of β-thalassemia: minihepcidin improves anemia and decreases iron overload
1600 16 1400 14 p = 0.001 p = 0.0003 1200 12
g/g) 1000 10 800 8
6 600 Liver iron ( iron Liver Hemoglobin (g/dl) Hemoglobin 4 400
2 200
0 0 WT TH3/solvent TH3/MH WT TH3/solvent TH3/MH Mouse/treatment Mouse/treatment
M004 (2.65 mg/kg) or solvent was administered 2x weekly SQ x 6 wks
C Casu, S. Rivella et al. and Merganser Biotech A search for erythroid factor suppressing hepcidin • Stimulated erythropoiesis in mice by phlebotomy (0.5 ml) or EPO injection (200 U) 4
phlebotomy 2 * p<0.05 EPO ** p<0.01 1 * ***p<0.001 * ** 0.5
mRNA expression *** *** 0.25 *** *** Hamp 0.125 Liver 0.062 CTRL 4h 9h 12h 15h 24h
• Gene chip expression profiling of the bone marrow to identify erythroid transcripts that 1) change before the hepcidin suppression and 2) encode secreted proteins Erythroferrone (Erfe) Erythroferrone (Fam132b, CTRP15)
TNFα-like
Polybasic cleavage sites?
Signal sequence
Collagen stalk GPP-rich
• 50 kD glycoprotein highly expressed in erythropoietin-stimulated erythroblasts Erfe knockout mice do not suppress hepcidin in response to bleeding • Erfe1+/+, +/- and -/- mice were phlebotomized (0.5 ml) Hepcidin mRNA Hemoglobin recovery
* p<0.05 ** p<0.01 *** p<0.001 Dose-response for mouse erythroferrone on hepatocytes
1.0 EC50 = 7 ng/ml ~ 140 pM 0.8
0.6
0.4
Fractional response Fractional 0.2
0.0
0 1 3 10 30 100 300 1000 Conc ng/ml mErfe (24-340) Mouse model of β-thalassemia: Ablation of Erfe reverses hepcidin suppression and improves iron overload
Hepcidin Liver iron 200 *** 300 *** *** *** * 150
200 100
50 100 serum hepcidin (ng/mL)
0 g non-heme iron / g liver wet weight 0 WT WT Th3/+ Th3/+ Erfe-/-Th3/+ Erfe-/-Th3/+ Development of new treatments
• Prevent iron overload and improve erythropoiesis in β-thalassemia – minihepcidins (hepcidin agonists) – erythroferrone antagonists • Release sequestered iron in anemia of inflammation and anemia of chronic kidney disease – hepcidin antagonists – erythroferrone agonists • UCLA licensed the intellectual property related to minihepcidin and erythroferrone to two startup companies for drug development Key contributors
Ella Nemeth: Piotr Ruchala: Everything Minihepcidin design
Airie Kim: León Kautz: Mouse models Erythroferrone of anemia of inflammation