THE ROLE OF HEMOJUVELIN IN IRON DEFICIENCY AND OVERLOAD

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1 THE ROLE OF HEMOJUVELIN IN IRON DEFICIENCY AND OVERLOAD Clara Camaschella Vita-Salute University and San Raffaele Scientific Institute Milan, Italy EHA ESH joint Workshop Cascais, Portugal, April 16-18, 2010

2 Hemojuvelin a multifaceted protein Cloned as the gene of juvenile hemochromatosis BMP co-receptor in iron-dependent hepcidin activation Suppressor of hepcidin transcription Substrate target of the serine protease TMPRSS6/matriptase-2 Neogenin interactor

3 6 HFE Hemochromatosis: molecular genetics 1 TYPE GENE OMIM PHENOTYPE HJV Type 1 HFE classic 19 Type 2a 2b HJV HAMP # juvenile 7 HAMP Type 3 TFR2 # intermediate Type 4 SLC40A1 # ferroportin disease TFR2 Type 1-3: autosomal recessive Type 4: autosomal dominant 2 SLC40A1

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5 Serum hepcidin dosage by ELISA (Ganz et al. Blood 2008;112: ) Hjv-/- mice Hjv -/-: iron overload Low levels of liver hepcidin RNA (Huang et al JCI 2005; Niederekofer et al JCI 2005)

6 Role of hemojuvelin in hepcidin regulation HJV: homologous to Repulsive Guidance Molecules (RGMa and RGMb) espressed in central nervous system and involved in axon guidance HJV/RGMc: expressed in liver, skeletal muscle, heart m-hjv: GPI-anchored, BMP co-receptor, hepcidin activator s-hjv: released form, decoy molecule, hepcidin inhibitor both in vitro and in vivo

7 Hepcidin upregulation Hemochromatosis proteins Inflammatory Pathway LPS *BMP6 *HJV *HFE *TFR2 BMPR IL-6 IL-1 IL-6R Macrophage /MAPK SMAD1/5/8P? STAT3 Hepatocyte 5 SMAD1/5/8 *SMAD4 P *HAMP * Indicates proteins whose inactivation causes iron overload

8 Hemojuvelin mutants C80R S85P G99V C119F A168D F170S D172E W191C G320V R326X R335Q N SP RGD GDPH Partial vwf type D RNRR GPI C RGD JH associated mutants GDPH GDPH mutants Artificial mutants RNRR Furin cleavage site mutants Why HJV mutants do not activate hepcidin?

9 Defective targeting of HJV to plasma membrane is a common pathogenetic mechanism in JH G99V C119F W191C F170S G320V TAA ATG 5 3 anti-hjv ER Golgi PM Anti-HJV Anti-BMP6 WT G99V (Silvestri L. et al. Blood 2007) (Pagani A. et al, Haematologica 2008 and unpublished)

10 Role of hemojuvelin in hepcidin regulation HJV: homologous to Repulsive Guidance Molecules (RGMa and RGMb) espressed in central nervous system and involved in axon guidance HJV/RGMc: expressed in liver, skeletal muscle, heart m-hjv: GPI-anchored, BMP co-receptor, hepcidin activator (and neogenin ligand) s-hjv: released form, decoy molecule, hepcidin inhibitor both in vitro and in vivo

11 s-hjv is iron regulated and regulates hepcidin (Lin et al. Blood 2005;106:2884-9) s-hjv binds BMP 2,4,5,6 Chronic s-hjv administration (3 weeks) induces iron overload and hepcidin suppression in mice (Babitt et al, 2007)

12 s-hjv is produced by HJV mutants in culture media _ + _ + _ + _ + _ + _ + medium total lysate + = 50 µμ FAC (Silvestri et al. Blood 2007;109: ) We dispute that s-hjv is produced by shedding of membrane hemojuvelin, as proposed by other Authors

13 No Caption Found s-hjv is produced by Furin cleavage h-hjv m-hjv h-rgma m-rgma h-rgmb m-rgmb 335 * (Silvestri, L. et al. Blood 2008;111: )

14 s-hjv is produced in iron deficiency/hypoxia (Silvestri et al, Blood 2008)

15 Physiological role of s-hjv? HJV is highly expressed in skeletal muscles, which do not express hepcidin Signal of muscle iron deficiency (?) in order to modulate hepcidin activation?

16 Hepcidin downregulated by multiple signals Hypoxia, iron deficiency Ineffective erythropoiesis s-hjv HFE HJV TFR2 TWSG1 BMPR GDF15? 5 HIF-1α HRE hepcidin

17 TMPRSS6/Matriptase-2:a novel hepcidin inhibitor A transmembrane serine protease (TTPS family) The main hepcidin inhibitor TMPRSS6 deficiency causes a genetic iron deficiency disorder in mice and IRIDA in humans

18 The Mask Mouse Iron-deficiency anemia, truncal alopecia high hepcidin, reduced iron absorption (Du et al, Science 2008)

19 The Tmprss6 -/- Mouse (Folgueras et al, Blood 2008)

20 TMPRSS6/Matriptase-2 TMPRSS6 gene: 18 exons on human chromosome 22 RNA expression: liver (kidney, olfactory epithelium) Protein: 811 amino acid type II transmembrane serine protease Matriptase-2 synthesized as an inactive zymogen (TTPS family: enteropeptidase, hepsin, corin, matriptase 1 ) N TM SEA CUB CUB L L L SERINE PROTEASE C

21 IRIDA: Clinical and laboratory features Autosomal recessive disorder (MIM #206200) that leads to microcytic hypochromic anemia Extremely low iron and transferrin saturation from infancy Normal serum ferritin High/normal (serum, urinary) hepcidin levels Unresponsive to oral and partially responsive to i.v. iron Different TMPRSS6 mutations, both null and missense, most private

22 Function and substrate of matriptase-2 The hypothesis Matriptase-2 either activates by cleavage an hepcidin inhibitor or inhibits by cleavage an hepcidin activator The HJV hypothesis m-hjv: is a BMP coreceptor and an hepcidin activator s-hjv: is a decoy molecule, inhibitor of hepcidin transcription in vitro and in vivo

23 Matriptase-2 cleaves membrane-hjv (Silvestri L. et al, Cell Met 2008;8:502-11)

24 Genetic interaction TMPRSS6-HJV IDA Iron overload Hb serum iron hepcidin X normal Hb serum iron hepcidin Mask Hjv-/- normal Hb serum iron hepcidin Iron overload Mask/Hjv-/- (Truska et al, BJH :571-81; Finberg et al Blood online 2010)

25 Model of hepcidin regulation by TMPRSS6 m-hjv BMP m-hjv BMP m-hjv BMP TMPRSS6 BMPR TMPRSS6 BMPR TMPRSS6 BMPR SMADs SMADs SMADs HAMP serum iron HAMP serum iron HAMP (undue) serum iron (undue) normal erythropoiesis IRIDA Juvenile Hemochromatosis

26 HJV the substrate of TMPRSS6/ Matriptase-2 No direct evidence in vivo biochemical evidence in vitro genetic indirect evidence from knock out mice models

27 TMPRSS6 mutants W247fs A118D S304L Y335X Y393X G442R E461fs E486D D521N E522K Mask R599X A605fs K636fs L674F P686fs R774C Y141C L166fs I212T Q229fs R271Q C510S S561X S570fs Biochemical and functional analysis of IRIDA-associated TMPRSS6 mutants show both reduction of hepcidin activation and of hemojuvelin cleavage (Silvestri et al, Cell Met 2008, Ramsay et al, Hum Mol Genet, 2009; Silvestri et al, Blood 2009, De Falco et al, Hum Mut 2010)

28 Hemojuvelin cleavage HJV undergo several distinct cleavage processes: autocatalitic cleavage at GDPH motif to produce the mature form (m-hjv) intracellular furin cleavage to produce s-hjv (muscle cells? liver?) TMPRSS6/matriptase-2 surface cleavage to produce small HJV fragments (hepatocytes, other cells?)

29 Hemojuvelin cleavage processes

30 The central role of HJV in iron metabolism The function of m-hjv as BMP-coreceptor is essential for hepcidin activation (JH and KO mice) Cleavage of m-hjv is essential for hepcidin inhibition (IRIDA, Mask mouse) in iron deficiency s-hjv (and other inhibitors) do not compensate for the lack of matriptase-2 (role in iron deficiency?) Persistent lack of m-hjv in JH indicates that Matriptase-2 protease activity should be tightly controlled Modulation of the pathway may be relevant for innovative treatments of iron disorders

31 Acknowledgments Laura Silvestri, Alessia Pagani, Antonella Nai Ivana De Domenico Jerry Kaplan Utah University Carlos Lopez Otin Oviedo University Paolo Arosio Brescia University e-rare Ministero dell'università e della Ricerca (PRIN) - Rome