Metabolismo del ferro in condizioni

normali e patologiche

Clara Camaschella

Università Vita-Salute e IRCCS San Raffaele, Milano

Simposio SIES41°Congresso Nazionale SIE - Bologna 14-17 ottobre 2007

Metabolismo del ferro in condizioni

normali e patologiche

Clara Camaschella

Università Vita-Salute e IRCCS San Raffaele, Milano

Simposio SIES41°Congresso Nazionale SIE - Bologna 14-17 ottobre 2007

Total body iron: ~ 4 g

Andrews, NEJM, 1999

Compartments:

functionaltransportstorage

NTBI appears when plasmatic Fe > binding capacity of transferrin

(at saturations > 60) is uptaken by cells and has great cell toxicity%)

Non-transferrin bound iron (NTBI)

Fe2Tf

Tf Fe

+

ROS

Danno

mitocondriale Danno

del DNAPerossidazione

lipidica

Danno delle

proteine

Lisosomi

Regulation of iron homeostasis

Cellular regulation (IRE-IRPs)

Systemic regulation (hepcidin-ferroportin)

Tissue regulation (??)

AAAA

5’

AAAA

5’AAAA

5

’

5’AAAA

UNSTABLE

STABILE

HIGH TRANSLATION

NO TRANSLATION

transferrin receptor mRNA ferritin mRNA

IRP1,IRP2

low

affinity

form

high

affinity

form

- IRON+ IRON

IRE-IRP POST-TRANSCRIPTIONAL REGULATION SYSTEM

Hepcidin: the key iron hormone

Struttura della proteina:8 cysteines: hairpin structure

(antimicrobial peptide)

expression: liver, skeletal muscle, heart

Acute phase reactant

Dosage: Prohepcidin (serum) 25-Aa peptide (serum, urine)

Liver peptide secreted as a precursor of 84 aminoacids.Active peptide (25 C-terminal aa) cleaved by a furin- like protein

Ganz, Blood 2003

Hepcidin: a a keykey ironiron regulatorregulator in in micemice

Hepc Hepc

Iron overload Iron deficiency

(Nicolas et al, PNAS 2001Lesbordes-Brion et al, Blood 2006)

(Nicolas et al, PNAS 2002)

Hepcidin: a a keykey ironiron regulatorregulator in in humanshumans

(Roetto et al Nat Genet 2003)

Transfection with

FPN-GFP Cell surface

FPN-GFP + InternalizationHEPCIDIN

Lysosomal degradation

(Nemeth et al,Science 2004))

Hepcidin regulates iron efflux by binding to ferroportin and inducing its internalization and lisosomal degradation

HEK293 cells

Iron-Hepcidin-FPN circuitry

MacrophageMacrophageEnterocyteEnterocyte

Hepcidin

Fe Inflammation (IL-6)

FeFe

FPN FPN

Iron-HEPC-FPN circuitry

FeFe

Hepcidin

erythroid expansion

FPN

EnterocyteEnterocyte MacrophageMacrophage

FPN

Fe,

Iron metabolism in genetic iron overload

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

skinskin

jointsjointspancreaspancreas

heartheartDietaryDietary ironiron

pituitary

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%)

¬ Autosomal recessive - rare

¬ Early onset (II-III decades)

¬ Both sexes

¬ Severe iron overload

¬ Progressive disease

¬ Mutations of hepcidin

or hemojuvelin

Juvenile hemochromatosis: type 2

liver

heart

pituitary

10

20

30

40

50

60

70

HFE JH

anni

• HH caused by mutations in HFE, transferrinreceptor 2, hemojuvelin or hepcidin gene

HEPCIDIN ANDHEREDITARY HEMOCHROMATOSIS

• Unifying characteristic – hepcidin deficiency

0

20

40

60

80

100

120

hep

cid

in (

ng

/mg

cre

ati

nin

e)

*

*

n= 105 33 10 7 3

Controls HFE TfR2 HJV Hepc

*

HJV: coreceptor for BMP

(Wang, Cell Metab 2005; 2:399-409)

Smad4 liver conditionla knock out developsiron overload

Liver iron accumulation

Great reduction of hepcidin expression

Hemojuvelin (HJV)

N CSP

RG

D

Partial vWF type D GPI

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)

HJV belongs to the Repulsive Guidance Molecules family

Defective targeting of HJV mutants to PM

0

10

20

30

40

50

60

1

% c

ell

surf

ace H

JV

*

*

*

*

WT

G99V

C119F

F170S

W19

1C

G320V

3’

ATG TAA

5’

G320V

G99V

R326X

F170S

W191CC119F

G99V

C119F

F170S

W19

1C

G320V

WT

mock

47.5

32.5

(Silvestri et al, Blood 2007)

Hepatocyte

hepcidin5’

Regulatory Complexfor Iron Homeostasis

HFETFR2

HJV

BMPR

SMAD4

Upregulation of hepcidin by hemochromatosis proteins

Hepatocyte

hepcidin5’

Macrophage

HfeTfr2

Hjv

BMPR

SMAD4

IL-6R

IL-6IL-1

LPS

STAT3?

Upregulation of hepcidin by inflammation

IL-1liver

IL-6

hepcidin

Hepcidin in inflammation

LPS

Reduced iron absorption

Reduced iron recycling

Iron retention in macrophages

(all features of ACD)IL-6 causes hypoferremia ininflammation inducing hepcidin synthesis

(Nemeth et al J Clin Invest 2004;113:1271-6)

Transcriptional regulation of hepcidin

Upregulation Downregulation

Hepcidin promoter

HJVHFETFR2FeIL-6

HypoxiaAnemiaIron deficiencyErythroid expansion

+

s-HJV is decreased by iron addition in both wt and mutants

W191C

47.5 -

47.5 -

G99VC119F

F170SG320V

WT

_+

+ = addition of 50 μMol FAC

_+

_+

_+

_+

_+

medium

total lysate

s-HJV is decreased by iron addition and increased by iron deficiency

(Silvestri et al, Blood 2007)

(Lin et al, Blood 2005)

Soluble HJV (s-HJV)

C

47.5

32.5

47.5

32.5

s-HJV

c-HJV

*

-tubulin

mockHJV

HJV+CMK

HJVR335Q

47.5

DHJVfurin

++-

s-HJV

c-HJV

47.5

32.5

+

47.5

furin83

--+ furinW547R

+ +

---

-tubulin47.5

N CSP

RG

D

Partial vWF type D GPI

332-335RNRR

s-HJV is produced by furin cleavage

(Silvestri et al, Blood 2007 online)

s-HJV is produced in iron deficiencyand hypoxia

(Silvestri et al, Blood 2007 on line)

A dual role of hemojuvelin

m-HJV: important in iron overloadferro. The absence of m-HJVcharacterizes juvenile hemochromatosis: loss of BMP signalling?

s-HJV: important in irondeficiency/hypoxia, not in juvenilehemochromatosis. Possible signal fromhypoxic muscles

(Silvestri et al, Blood 2007)(Lin et al, Blood 2005)

Hepcidin in secondary iron overload

“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

normal TI TM

10

100

1000

2787575178Ferritin

sTfR

Hb

% Tf sat 957933

108.414.6

12471.4

Hepcidin in thalassemia

(Origa et al, Haematologica 2007;92:583-8)

Tissue

Iron

Bone

marrow

Liver

Plasma

Iron

hepcidin

Erythropoietic signal

Iron signal

HEPCIDIN REGULATION BY ANEMIA

A candidate “erythroid regulator”

Tanno et al, High levels of GDF15 in thalassemia suppress expression of the iron

regulatory protein hepcidin. Nat Med. 2007;13:1096-101

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)

Genetic disorders affecting different steps of the iron regulatory loop

Camaschella, Blood 2005;106:3710-7

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

Hypo-transferrinemia

Hpx miceSimilar phenotypeSplicing mutations of TFLiver hepcidin RNA low/absent

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 transporterErythroblasts: endosomal transferrin cycleMacrophage: iron recycling

FPNeph

DMT1

dcytb

absorption

1DMTI

utilization

Animal models

mk mouse and Belgrade ratsevere microcytic hypochromic iron-deficient anemiadue to the same (G185R) DMT1 mutation

Patients with DMT1 mutations

(Priwitzerova et al Blood 2004, Mims et al Blood 2005Iolascon et al Blood 2005Beaumont et al, Blood 2006)

microcytic hypochromic anemia from birth and liver iron overload

(Andrews, NEJM, 1999)

Reduced iron supply to the marrow

Suppression of hepcidinproduction

Increased duodenal iron absorption

Sideroblastic anemias

Perl’s staining

Anti-MT-ferritin

(Courtesy of R. Invernizzi, Pavia)

Mitochondrial iron metabolism

(Modified from Blood 105;1867-1874, 2005)

Heme

H-ferritin

L-ferritin

M-Aconitase

ALAS2

Ferroportin

TfR

DMT1-IRE

Others?

IRPs regulation and IRP target mRNA

Target

genes

(Blood 2007;110:1353-8)

A complex case

HUMAN DEFICIENCY OF GLRX5

Homozygous splicing mutation of GLRX5

GLRX5 deficiency reduces Fe/S cluster andcauses hyperactivity of IRP1 with ALAS2repression and heme reduction

Transfusional iron overload worsens anemia

Iron chelation by DFO ameliorates anemia

(Blood 2007;110:1353-8)

Acknowledgments

University Vita-Salute IRCCS San Raffaele

Sonia Levi Alessandro Campanella

CEINGE Naples University of Torino

Achille Iolascon Antonella Roetto

Maria D’Apolito Roberta Merlini

University of Brescia

Paolo Arosio

Alessia PaganiLaura Silvestri