Metabolismo del ferro in condizioni normali e patologiche

Transcription

1 Metabolismo del ferro in condizioni normali e patologiche Clara Camaschella Università Vita-Salute e IRCCS San Raffaele, Milano Simposio SIES 41 Congresso Nazionale SIE - Bologna ottobre 2007

2 Metabolismo del ferro in condizioni normali e patologiche Clara Camaschella Università Vita-Salute e IRCCS San Raffaele, Milano Simposio SIES 41 Congresso Nazionale SIE - Bologna ottobre 2007

3 Total body iron: ~ 4 g Compartments: functional transport storage Andrews, NEJM, 1999

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6 Non-transferrin bound iron (NTBI) Tf Fe + Fe 2 Tf NTBI appears when plasmatic Fe > binding capacity of transferrin (at saturations > 60) is uptaken by cells and has great cell toxicity%) ROS Danno mitocondriale Perossidazione lipidica Danno del DNA Danno delle proteine Lisosomi

7 Regulation of iron homeostasis Cellular regulation (IRE-IRPs) Systemic regulation (hepcidinferroportin) Tissue regulation (??)

8 IRE-IRP POST-TRANSCRIPTIONAL REGULATION SYSTEM transferrin receptor mrna ferritin mrna UNSTABLE IRP1,IRP2 HIGH TRANSLATION 5 AAAA low affinity form 5 AAAA + IRON - IRON STABILE NO TRANSLATION 5 high affinity form 5 AAAA AAAA

9 Hepcidin: the key iron hormone Liver peptide secreted as a precursor of 84 aminoacids. Active peptide (25 C-terminal aa) cleaved by a furin- like protein Struttura della proteina: 8 cysteines: hairpin structure (antimicrobial peptide) expression: liver, skeletal muscle, heart Acute phase reactant Ganz, Blood 2003 Dosage: Prohepcidin (serum) 25-Aa peptide (serum, urine)

10 Hepcidin: a key iron regulator in mice Hepc Hepc Iron overload Iron deficiency (Nicolas et al, PNAS 2001 Lesbordes-Brion et al, Blood 2006) (Nicolas et al, PNAS 2002)

11 Hepcidin: a key iron regulator in humans (Roetto et al Nat Genet 2003)

12 Hepcidin regulates iron efflux by binding to ferroportin and inducing its internalization and lisosomal degradation HEK293 cells Transfection with FPN-GFP Cell surface FPN-GFP + Internalization HEPCIDIN Lysosomal degradation (Nemeth et al,science 2004))

13 Iron-Hepcidin-FPN circuitry Fe Inflammation (IL-6) Hepcidin FPN FPN Enterocyte Fe Macrophage

14 Iron-HEPC-FPN circuitry Fe, erythroid expansion Hepcidin FPN FPN Enterocyte Fe Macrophage

15 Iron metabolism in genetic iron overload

16 HEMOCHROMATOSIS: pathogenesis The genetic defect causes inappropriately high intestinal iron absorption and macrophage iron recycling leading to: increased transferrin saturation increased serum ferritin iron accumulation in parenchymal organs iron toxicity and organ failure pituitary skin heart Dietary iron pancreas joints

17 Hemochromatosis type 1 Most common form Autosomal recessive - Late onset (40-60 years) Prevalent expression in male Increased intestinal iron absorption Iron storage in hepatocytes HFE mutations (C282Y mutation at the homozygous state 60->90%)

18 Juvenile hemochromatosis: type 2 Autosomal recessive - rare Early onset (II-III decades) Both sexes anni HFE JH Severe iron overload Progressive disease liver Mutations of hepcidin or hemojuvelin heart pituitary

19 HEPCIDIN AND HEREDITARY HEMOCHROMATOSIS HH caused by mutations in HFE, transferrin receptor 2, hemojuvelin or hepcidin gene Unifying characteristic hepcidin deficiency hepcidin (ng/mg creatinine) * * Controls HFE TfR2 HJV Hepc n= *

20 HJV: coreceptor for BMP

21 Smad4 liver conditionla knock out develops iron overload Liver iron accumulation Great reduction of hepcidin expression (Wang, Cell Metab 2005; 2: )

22 Hemojuvelin (HJV) HJV belongs to the Repulsive Guidance Molecules family N SP RGD Partial vwf type D GPI C RGMa and RGMb: - expressed in the central nervous system - involved in axon guidance RGMc/HJV: - expressed in liver, skeletal muscle, heart - involved in iron metabolism - membran form (m-hjv) - soluble form (s-hjv)

23 % cell surface HJV WT G99V C119F F170S W191C G320V Defective targeting of HJV mutants to PM R326X G99V C119F W191C F170S G320V ATG TAA * * * * (Silvestri et al, Blood 2007) mock WT G99V C119F F170S W191C G320V

24 Upregulation of hepcidin by hemochromatosis proteins Regulatory Complex for Iron Homeostasis HJV HFE TFR2 BMPR Hepatocyte SMAD4 5 hepcidin

25 Upregulation of hepcidin by inflammation LPS Macrophage Hfe Tfr2 Hjv IL-6 IL-1 BMPR IL-6R SMAD4? STAT3 Hepatocyte 5 hepcidin

26 Hepcidin in inflammation LPS liver IL-6 IL-1 hepcidin IL-6 causes hypoferremia in inflammation inducing hepcidin synthesis Reduced iron absorption Reduced iron recycling Iron retention in macrophages (all features of ACD) (Nemeth et al J Clin Invest 2004;113:1271-6)

27 Transcriptional regulation of hepcidin Upregulation HJV HFE TFR2 Fe IL-6 Downregulation Hypoxia Anemia Iron deficiency Erythroid expansion + Hepcidin promoter

28 Soluble HJV (s-hjv) s-hjv is decreased by iron addition and increased by iron deficiency (Lin et al, Blood 2005) s-hjv is decreased by iron addition in both wt and mutants _ WT + _ G99V + _ C119F + _ F170S + _ W191C + _ G320V medium total lysate + = addition of 50 μmol FAC (Silvestri et al, Blood 2007)

29 s-hjv is produced by furin cleavage N SP RGD Partial vwf type D RNRR GPI C C mock HJV HJV+CMK HJV R335Q s-hjv * c-hjv D furinw547r furin + + HJV s-hjv c-hjv tubulin 47.5 furin -tubulin (Silvestri et al, Blood 2007 online)

30 s-hjv is produced in iron deficiency and hypoxia (Silvestri et al, Blood 2007 on line)

31 A dual role of hemojuvelin m-hjv: important in iron overload ferro. The absence of m-hjv characterizes juvenile hemochromatosis : loss of BMP signalling? s-hjv: important in iron deficiency/hypoxia, not in juvenile hemochromatosis. Possible signal from hypoxic muscles (Silvestri et al, Blood 2007) (Lin et al, Blood 2005)

32 Hepcidin in secondary iron overload

33 Hepcidin in Iron loading anemias Inherited anemias characterized by ineffective Erythropoiesis and increased intestinal iron absorption leading to secondary iron overload thalassemia, CDA sideroblastic anemia Reduction of hepcidin in the untransfused cases is a general mechanism of iron overload in these conditions

34 Hepcidin in thalassemia normal TI TM % Tf sat Ferritin Hb stfr (Origa et al, Haematologica 2007;92:583-8)

35 HEPCIDIN REGULATION BY ANEMIA Iron signal Liver Plasma Iron Tissue Iron hepcidin Erythropoietic signal Bone marrow

36 A candidate erythroid regulator Tanno et al, High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med. 2007;13: Increased expression and secretion during erythroblast maturation of Growth differentiation factor 15 (GDF15), a member of the TGF- superfamily Elevated GDF15 serum levels in -thalassemia syndromes (mean 66,000 +/- 9,600 pg/ml; P < 0.05 vs normals) Suppression of hepcidin mrna expression in primary human hepatocytes by thalassemia serum (Tanno et al, Nat Med, 2007)

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39 Genetic disorders affecting different steps of the iron regulatory loop Camaschella, Blood 2005;106:3710-7

40 Hypo-transferrinemia Autosomal recessive, extremely rare First description 1961 Plasma transferrin nearly absent Severe microcytic anemia since birth Liver iron overload Responsive to plasma (TF) infusions Low urinary hepcidin Hpx mice Similar phenotype Splicing mutations of TF Liver hepcidin RNA low/absent

41 New rare disorders of iron utilization: DMT1 deficiency DMT1: Transporter of divalent metal cations (Mn 2+ Cu 2+ Zn 2+ Fe 2+ ) Duodenal cell: luminal non heme iron transporter Erythroblasts: endosomal transferrin cycle Macrophage: iron recycling DMT1 dcytb FPN eph DMTI 1 absorption utilization

42 Animal models mk mouse and Belgrade rat severe microcytic hypochromic iron-deficient anemia due to the same (G185R) DMT1 mutation Patients with DMT1 mutations microcytic hypochromic anemia from birth and liver iron overload (Priwitzerova et al Blood 2004, Mims et al Blood 2005 Iolascon et al Blood 2005 Beaumont et al, Blood 2006)

43 Reduced iron supply to the marrow Suppression of hepcidin production Increased duodenal iron absorption (Andrews, NEJM, 1999)

44 Sideroblastic anemias Perl s staining Anti-MT-ferritin (Courtesy of R. Invernizzi, Pavia)

45 Mitochondrial iron metabolism Heme (Modified from Blood 105; , 2005)

46 IRPs regulation and IRP target mrna Target genes H-ferritin L-ferritin M-Aconitase ALAS2 Ferroportin TfR DMT1-IRE Others?

47 A complex case (Blood 2007;110:1353-8)

48 HUMAN DEFICIENCY OF GLRX5 Homozygous splicing mutation of GLRX5 GLRX5 deficiency reduces Fe/S cluster and causes hyperactivity of IRP1 with ALAS2 repression and heme reduction Transfusional iron overload worsens anemia Iron chelation by DFO ameliorates anemia (Blood 2007;110:1353-8)

49 Acknowledgments University Vita-Salute IRCCS San Raffaele Laura Silvestri Alessia Pagani Sonia Levi Alessandro Campanella CEINGE Naples Achille Iolascon Maria D Apolito University of Torino Antonella Roetto Roberta Merlini University of Brescia Paolo Arosio