Interplay of hepatocellular and hematopoietic system (iron storage, anemia, Epo)
Participating groups:
Martina Muckenthaler (PhD); Dept of Pediatric Oncology, Hematology and Immunology; University of
Heidelberg; Im Neuenheimer Feld 153; 69120 Heidelberg
Tel.: 0049 - 06221 566923; e-mail: martina.muckenthaler@med.uni-heidelberg.de
Matthias Hentze (Dr. med.) EMBL, European Molecular Biology Laboratory; Meyerhofstrasse 1, D-69117;
Heidelberg, Germany
Tel.: +49-6221-387 501 (office); e-mail: hentze@embl.de
Jens Georg Reich (Prof. Dr.); Humboldt University (Charité); Max-Delbrück-Center for Molecular
Medicine; 13092 Berlin
Tel.:
e-mail: reich@mdc-berlin.de
Oliver Leismann (PhD) Novartis Pharma
Ursula Klingmüller – bitte Addresse einfügen
State of the art, and own contributions (1/2 page)
Balancing systemic iron levels within narrow limits is critical for human health: diseases of iron deficiency
and overload belong to the most frequent disorders world-wide. Regulation of systemic iron homeostasis
evolved to maintain a plasma iron concentration that secures adequate supplies while preventing organ iron
overload. The regulatory system responds to signals from pathways that consume iron (e.g. erythropoiesis)
and transmits signals to cells that supply iron (e.g. duodenal enterocytes which absorb dietary iron;
macrophages which recycle iron from effete erythrocytes, and hepatocytes that store iron). The small hepatic
peptide hormone Hepcidin synchronizes these systemic iron fluxes by binding to the iron export channel
ferroportin located on the surface of macrophages, hepatocytes and intestinal enterocytes to cause its
internalization and proteolysis. In accordance with this mechanism chronically elevated hepcidin levels (e.g.
in the anemia of chronic diseases; ACD) cause systemic iron deficiency and inappropriately low hepcidin
levels (e.g. in hereditary hemochromatosis; HH) result in iron overload.
It is widely accepted that erythropioetic signals (e.g. from the bone marrow) to the liver regulate systemic
iron availability according to the demand of the erythron. In disorders that are hallmarked by mutations in
genes that impair the generation of mature erythrocytes (e.g. mutations in the globin genes in the
thalassemias) the same regulatory circuit seems to signal insufficient iron availability (despite full liver iron
stores) and as a consequence hepcidin expression is decreased, iron uptake increased and secondary hepatic
iron overload develops. Neither the erythropoietic regulator nor the hepatic pathways that control hepcidin
expression in response to erythropoietic activity have been identified so far.
Description of planned work for five years, including clearly formulated milestones after three
years (maximum 1.5 page)
The overall aim of this project is to characterize the general response of the hepatic iron regulatory
network and specifically, of the signalling pathways that regulate hepcidin mRNA expression
(TGFß/BMP/SMAD, JAK/STAT and others) to increased erythropoietic activity.
Experimentally, we will stimulate erythropoietic activity in mice by (1) phlebotomy (bleeding) or
(2) by treatment with erythropoietin (Epo). Two time points after treatment will be further
investigated. In a first approach, we will analyze the success of these treatments by investigating
haematological parameters (Hb, MCV, reticulocyte counts) and serum iron parameters and correlate
these findings with the activity of the major hepatic signalling pathways that also regulate hepcidin
expression (TGFß/BMP/SMAD and JAK/STAT pathways). To this end will investigate
SMAD1/5/8; SMAD2, SMAD3 and SMAD4 or STAT3 and STAT5 by western blotting for total
protein amounts and phosphorylated forms of the proteins
In a second step, we will perform comprehensive gene expression profiling/(possibly also
proteomic analysis in collaboration with xxx) of the livers of phlebotomized and Epo-treated mice
and compare the differentially expressed genes with functional data from a genome-wide RNAi
screen for hepatic hepcidin regulators that are available in the lab. This experimental approach will
allow a detailed and comprehensive analysis of hepatic pathways that regulate hepcidin expression
in response to increased erythropoietic activity. Further functional characterization of the identified
genes and signalling pathways in primary hepatocytes will yield a detailed molecular understanding
of pathways responsible for the cross-talk between the erythron and the liver. These data will
provide unique information for the modelling of these important pathways.
Third, we will treat mice with primary iron overload due to HFE-deficiency with EPO or
phlebotomy to observe how signalling is affected by the Hfe-deficiency and/or the liver iron
overload.
In a second experimental approach we will make use of previously established mouse lines with
global or cell type-specific IRP1 and/or IRP2 ablations. IRP2-deficient mice exhibit microcytic
anemia, which can be explained by an erythroid-cell autonomous response. Further important cell
type specific functions of IRPs in regulating iron absorption, iron retention and erythropoietic
activity have been unravelled. Existing mouse models now allow to assess the role of this
regulatory system specifically in hepatocytes, and to combine experimentation and modelling to
define the cross-talk between the hepcidin and the IRE/IRP system and erythropoietic stimulation
and IRP activity in the liver.
Specifically, we will make use of mice with (i) inducible hepatic IRP1 and/or IRP2 ablations and
(ii) inducible IRP1 overexpression in the liver.
In these mouse models we will
1) evaluate the response of the hepcidin system to hepatocytic IRP ablation or overexpression,
which is of particular interest considering that hepatocytes are the critical cell type for
systemic iron sensing and the systemic response via hepcidin.
2) Specific focus will be given to the hepatocytic response to increased erythropoietic activity
due to phlebotomy or EPO treatment to assess the contribution of the IRE/IRP regulatory
system to the regulation of hepcidin
Mathematical modelling of systemic iron homeostasis and liver regeneration
Based on previous work the Systems Biology aspect of iron metabolism will be pursued further, in
close interaction with experimental studies and with modelling in other projects of how increased
erythropoietic activity affects
 hepatocyte iron metabolism, under signalling influence of global iron status, with internal
feedback steering via IRP and Hfe-controlled regulation
 global structure of liver-body-interaction via hormonal signalling (hepcidin, erythropoetin).
The methodical aspects of this modelling project will be tightly coupled with and coordinated by
the methodical network of HepatoSys, with an ultimate aim at a global theory of liver in
physiological and pathological conditions.
Milestones to be reached after 3 years:
(1) Two mouse models hallmarked by increased erythropoietic activity are available (EPO
stimulation, phlebotomy model)
(2) The response of the TGFß/BMP/SMAD and JAK/STAT pathways is analyzed at 2 different
time points following erythropoietic stimulation.
(3) Hepatic gene expression profiles in EPO stimulated and phlebotomized mice are correlated
with data from the siRNA screen for hepcidin activators or repressors.
(4) The hepcidin response is analyzed in mice with hepatic IRP ablation and overexpression.
(5) EPO stimulation and phlebotomy is applied in mouse models with IRE/IRP impairment.
(6) Model how increased erythropoietic activity affects important liver specific pathways is
available.
Contribution to economic exploitation (¼ page)
The project will yield fundamental insight into how the liver responds to increased erythropoietic
stimulation. This knowledge may provide ….
Five major recent subject-related publications
Muckenthaler M., Roy CN, Custodio AO, Minana B, DeGraaf J, Montross LK, Andrews NC, Hentze MW. Regulatory
defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nature
Genetics 34:102-7 (2003).
Roy, C.N., Custodio, A.O., de Graaf, J., Schneider, S., Akpan, I., Montross, L.K., Sanchez, M. Gaudino, A., Hentze,
M.W., Andrews, N.C., Muckenthaler, M.U. An Hfe-dependent pathway mediates hyposideremia in response to
lipopolysaccaride-induced inflammation in mice. Nature Genetics 36:481-485 (2004).
Galy, B., S.M. Hölter, T. Klopstock, D. Ferring, L. Becker, S. Kaden, W. Wurst, H.-J. Gröne and M.W. Hentze. Iron
homeostasis in the brain: complete iron regulatory protein 2 deficiency without symptomatic neurodegeneration in the
mouse. Nature Genetics 38, 967-969, 2006.
Maja Vujić Spasić, Judit Kiss, Thomas Herrmann, Bruno Galy, Stefanie Martinache, Jens Stolte, Hermann-Josef Gröne,
Wolfgang Stremmel, Matthias W. Hentze and Martina U. Muckenthaler. Hfe acts in hepatocytes to prevent
hemochromatosis. Cell Metabolism 2008 Feb;7(2):173-8.
Galy, B., D. Ferring-Appel, , S. Kaden, H.-J. Gröne and M.W. Hentze. Iron regulatory proteins are essential for
intestinal function and control key iron absorption molecules in the duodenum. Cell Metab. 7, 79-85, 2008