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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