1. health and fitness
2. disease

INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
MEETING PROGRAM
nd
Ferrum Rex
2 Meeting of The Polish Iron Club
Institute of Genetics and Animal Breediing, PAS
Jastrzębiec, May 12-13, 2015
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Dear Colegues,
Following the maxim ut ferrum sinon utaris, obdicitur rubigine (iron not used
soon rusts) we organize a meeting that is yet another initiative taken up in the last 5
years whose aim it is to integrate the actions of biologists, doctors and pharmacologists
dealing in the various aspects of iron biology.
Despite visible progress in the popularization of this subject matter in the Polish
science milieu, iron as a biometal mainly attracts the attention of hematologists. We try
to change this state of affairs. The main motif of our Ferrum Rex meeting does not
indicate we want to establish the supremacy of iron over other biometals, even though
we could with no effort at all point to some royal attributes of iron in biology – the good
and the bad ones. Indeed, it is our intention to draw attention to iron’s unique place in
biology, but also to its participation in many biological processes, which is very well
illustrated by the abundance and great variety of research themes mentioned during our
meeting.
A special place during our meeting is dedicated to heme iron. Recent research
indicates the existence of an autonomous system of proteins participating in intracellular
and systemic transport of heme in mammals. It seems that understanding the
functioning of this system as well as finding molecular links between the turnover of
heme iron and the turnover of ion iron in an organism is one of the most important
scientific challenges for Iron Men.
Paweł Lipiński
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
ORGANIZING COMMITTEE
Paweł Lipiński
Rafał R. Starzyński
Agnieszka Styś
Ewa Smuda
Robert Staroń
Małgorzata Prokopiuk
Anna Gajowiak
CONTACT
Paweł Lipiński
694489 005
Rafał R. Starzyński
509 818 609
SPONSORS
Beckman Coulter Polska Sp. z.o.o.
Roche Diagnostics, POLAND
Institute of Genetics and Animal Breeding, PAS
Medpharm Polska
Supported by National Science Centre grant № 2012/05/E/NZ5/02126
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
PROGRAM SCHEDULE
Monday, May 11
16:00 REGISTRATION
Tuesday, May 12
9:00 OPENING
Jarosław Horbańczuk, Director of IGAB PAS, Jastrzębiec, Poland
Paweł Lipiński, IGAB PAS, Jastrzębiec, Poland, on behalf of the Organizing
Committee
9:10-12:45 HEME METABOLISM SESSION
(presentations of invited speakers: 30 min +10 min discussion)
9:10-9:50 Alicja Józkowicz, Jagiellonian University, Cracow, Poland
Through stress or not through stress: how heme oxygenase-1 affects
myogenesis
9:50-10:30 François Canonne-Hergaux, INSERM, Centre de Physiopathologie
Toulouse Purpan, Toulouse, France
Erythrophagocytosis and heme iron recycling by macrophages
10:30-10:45 COFFEE BREAK
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10:45-11:25 Jozsef Balla, University of Debrecen, Debrecen, Hungary
Heme-stress in vascular biology
11:25-12:05 Søren Moestrup, Aarhus University, Aarhus, Danmark
Receptors for heme and hemoglobin scavenging
12:05-12:45 Francesca Vinchi, Heidelberg University Medical Center, Heidelberg,
Germany
Regulation and dysregulation of iron and heme metabolism
12:45-14.15 LUNCH
(BANQUET HALL)
14:15-16:15 SESSION 1
Presentations of young Polish researchers (15 min + 5 min discussion)
14:15-14:35 Robert Staroń, Institute of Genetics and Animal Breeding PAS, Jastrzębiec
Regulation of the molecular mechanisms of heme absorption: studies
on piglets with iron deficiency anemia supplemented with heme iron.
14:35-14:55 Aleksandra Bednarz, Jagiellonian University, Kraków
Identification of reticuloendothelial cells in kidneys of young mice and
their plausible function in iron metabolism
14:55-15:15 Agata Szade, Jagiellonian University, Cracow, Poland
Cobalt propoporphyrin: unexpected effect on mobilization of
hematopoietic cell
15:15-15:35 Anna Woziwodzka, Intercollegiate Faculty of BiotechnologyUniversity of
Gdańsk and Medical University of Gdańsk, Gdańsk
Genetic background of iron homeostasis disturbances in chronic
hepatitis C patients
15:35-15:55 Katarzyna Sikorska, University of Gdańsk, Gdańsk
IL-28 polymorphisms and iron homeostasis disturbances in relation to
hepcidin expression in chronic hepatitis C: polish single center study
15:55-16:15 Artur Słomka, Collegium Medicum of Nicolaus Copernicus University,
Bydgoszcz
The impact of treatment for acute ischemic stroke on hepcidin levels
16:15-16:30 COFFEE BREAK
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16:30-16:45 Rafał Kochanowski, Roche Dignostics Poland
Roche Molecular Diagnostics and Sequencing
16:45-17:00 Izabela Sadowska-Bartosz, University of Rzeszów, Rzeszów
Modification of the deoxyribose test to detect strong iron chelators
17:00-17:20 Ewelina Łukaszyk, Medical University, Białystok, Poland.
Association of serum levels of FGF23 with iron status, inflammation
and kidney functionamong patients with early stages of chronic
kidney disease
17:20-17:40 Radosław Kuliński, Medical University of Warsaw, Warszawa,
Are the mechanisms of nigral cells neurodegeneration in typical and
atypical parkinsonism different—MRI and Mössbauer spectroscopy
studies
17:40-18:00 Agnieszka Styś, Institute of Genetics and Animal Breeding PAS,
Jastrzębiec
Overexpression of both non-mutated human SOD1 and mutated
SOD1G93A genes induces changes of iron metabolism genes in mice
18:00-18:20 Mateusz Ogórek, Jagiellonian University, Kraków
Key role of copper in spermatogenesis
18:30-19:30 CONCERT OF CLASSICAL MUSIC
(CONFERENCE ROOM)
19:30 GALLA DINNER
(BANQUET HALL)
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Wednesday, May 13
9:00-12:00 SESSION 2
Presentations of young Polish researchers (15 min + 5 min discussion)
9:00-9:20 Monika Kasztura, Wroclaw Medical University, Wroclaw
Influence of iron availability on the cell viability and the expression of
genes associated with intracellular iron metabolism within rat H9C2
cardiomyocytes and L6G8C5 myocytes cultured either in static
conditions or upon mechanical stretch
9:20-9:40 Magdalena Stugiewicz, Wroclaw Medical University, Wroclaw
Influence of hypoxia on the molecular machinery involved in iron
metabolism within rat H9C2 cardiomyocytes and L6G8C5 myocytes
cultured in differential iron availability either in static conditions or
upon mechanical stretch
9:40-10:00 Witold Nowak, Jagiellonian University, Kraków
Still alive: mesenchymal stromal cells are resistant to oxidative stress
despite the low level of heme oxygenases
.
10:00-10:20 Mateusz Manicki, Intercollegiate Faculty of Biotechnology, University of
Gdańsk and Medical University of Gdańsk, Gdańsk
Binding of Hsp70 chaperone Ssq1, and frataxin homologue Yfh1, to
the iron-sulfur cluster scaffold Isu1 protein is mutually exclusive
10:20-10:40 COFFEE BREAK
10:40-11:00 Jędrzej Antosiewicz, Medical University of Gdansk
Homocysteine impairs iron metabolism in HUVEC cells
11:00:11:20 Olga Pierzchała, Jagiellonian University, Kraków
Impact of the copper deficiency on the expression of CTR1 gene and
cellular localisation of CTR1 protein in mice with mutation in ATP7A
gene
11:20-11:40 Sabina Galiniak, University of Rzeszów, Rzeszów
Is iron chelation important in preventing glycation of bovine serum
albumin in vitro?
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11:40-12:00 Magdalena Zielińska-Dawidziak, Poznań University of Life Sciences
Why do results of in vivo and in vitro experiments on ferritin iron
absorption greatly differ?
12:00-CLOSING REMARKS
Paweł Lipiński, IGAB PAS, Jastrzębiec, Poland, on behalf of the Organizing
Committee
CONFERENCE VENUE MAP
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ORAL PRESENTATIONS / ABSTRACT
Through stress or not through stress: how heme oxygenase-1 affects myogenesis
1
1
1
1
1
1
M. Cieśla , P. Marona , M. Seczynska , M. Kozakowska , M. Jez , A. Szade , M.
1
1
1
2
2
1
Walawender , J. Stepniewski , W.N. Nowak , A. Urbanik , B. Kazanowska , J. Dulak ,
1
A. Józkowicz
1
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków
2
Department of Oncology, Hematology and Bone Marrow Transplantation, Wroclaw
Medical University, Wroclaw
Heme oxygenase-1 (HO-1) degrades heme to ferrous iron, carbon monoxide (CO)
and biliverdin. Apart from inhibition of oxidative stress and inflammation, HO-1 facilitates
angiogenesis and influences both cell differentiation and cancer development. Here we
investigated the role of HO-1 in myogenic differentiation of myoblast and
rhabdomyosarcoma.
We found that retroviral overexpression of HO-1 in C2C12 myoblast cell line impairs
myogenic differentiation. It also reduces the expression of DGCR8 (heme-regulated
dimeric protein, involved in miRNA processing) and inhibits production of myomirs,
including miR-206. Inhibitory effects of HO-1 on maturation of myoblasts is independent
of oxidative stress but relies mainly on CO-mediated decrease in c/EBPδ binding to
myoD promoter.
HO-1 inhibits also differentiation of rhabdomyosarcoma (RMS), the soft tissue
sarcoma characterized by abrogation of myogenic program. Two main subtypes of RMS
are distinguished – benign embryonal (eRMS) and aggressive alveolar (aRMS). HO-1
expression is elevated in cell lines or clinical primary tumors of aRMS phenotype and
HO-1 upregulation is induced in eRMS by forced expression of aRMS hallmark Pax3/7FoxO1 fusion oncogene. Incubation of RMS cells with SnPP increases expression of
myogenic differentiation markers (e.g. myogenin, miR-206) and inhibits expression of
Pax3/7-FoxO1 target genes (SDF-1, CXCR4, c-met, HGF). Differently than in
myoblatsts, these effects are independent of cEBPδ/myoD interaction. Instead, high
level of HO-1 in aRMS is associated with decreased generation of reactive oxygen
species (ROS) leading to reduced translocation of nuclear histone deacetylase-4
(HDAC-4) to cytoplasm, what results in repression of miR-206 production. Inhibition of
HO-1 with tin protoporphyrin (SnPP) leads to elevation of ROS, oxidation and
cytoplasmic translocation of HDAC-4, and de-repression of miR-206, what triggers
myogenic differentiation of RMS cells. Accordingly, inhibition of HO-1 in nude mice
reduces growth and vascularization of human RMS tumors, what is accompanied by
upregulated expression of miR-206. Like in myoblasts, high level of HO-1 reduces
expression of DGCR8 protein and decreases production of pre-miRNAs.
In summary, HO-1 inhibits myogenic differentiation both in myoblasts and RMS cells.
The molecular pathways are, however, distinct: independent of oxidative stress (in
myoblasts) or dependent on antioxidative properties of HO-1 (in rhabdomyosarcoma).
THE POLISH IRON CLUB
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Erythrophagocytosis and heme iron recycling
François Canonne-Hergaux
INSERM UMR 1043; CNRS UMR 5282; Université de Toulouse, UPS, Centre de
Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
Macrophages play a critical role on the maintenance of iron homeostasis through heme
iron recycling following phagocytosis and degradation of senescent red blood cells
(sRBC). Erythrocyte ageing is associated with appearance of several senescence
markers at their cell surface which allow the specific recognition and engulfment of
sRBC by macrophages. Such process known as erythrophagocytosis (EP) or
extravascular hemolysis occurs mainly in the spleen and the bone marrow and in a
lesser extent in the kupffer cells of the liver. Following EP, heme catabolism by heme
oxygenase1 (HO1) lead to the release of heme iron which isthen recycled to the plasma
by macrophages through ferroportin (Fpn), the only known mammalian iron exporter.
This recycling process is highly regulated and is critical for mammalian iron
homeostasis, participating when disturbed to the development of anemia or iron
overload diseases such as hemochromatosis. Using a model that mimics the process of
erythrophagocytosis including red blood cells ageing, recognition and engulfment by
macrophages, we clarified the different steps of heme iron recycling by macrophages.
Subcellular localization studies of various heme- and iron-related proteins during early
steps of EP suggest a model for heme catabolism occurring outside the
erythrophagosome, with heme transported into the cytosol through HRG1. Heme
derived from RBC then leads to a predominantly iron-independent transcriptional
activation of Fpn and HO1 genes. Iron released from heme catabolism subsequently
strongly stimulates Fpn mRNA and protein expression through a post-transcriptional
regulation. Such increase of Fpn expression isassociated with accumulation of the iron
transporter at the plasma membrane. At the cell surface, Fpn is enriched in lipid
raftsmicrodomaines(cholesterol rich membrane compartments) which are important for
its regulation by hepcidin, a small peptide considered as the major hormonal regulator of
iron homeostasis. Some oxidases likely stabilize Fpn at the cell surface and participate
to the export of iron outside the macrophages. Interestingly, both hemeand iron can
inducethe presence of HO1 protein in lipid raftsand partialcolocalisationof HO1 with the
transporter Fpn at the cell surface. Integrating all of these observations, a model of
heme iron recycling in macrophages will be discussed.
THE POLISH IRON CLUB
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Heme-stress in vascular biology
József Balla
Department of Medicine, Division of Nephrology, Faculty of Medicine, University of
Debrecen, Debrecen, Hungary.
MTA-DE Vascular Biology, Thrombosis and Hemostasis Research Group, Hungarian
Academy of Sciences, Debrecen, Hungary.
Iron-derived reactive oxygen species are involved in the pathogenesis of
numerous vascular diseases. One abundant source of redox active iron is heme, which
is inherently dangerous when it escapes from its physiologic sites. In this review we
present the nature of heme-mediated vascular damage and of the strategies by which
vascular cells manages to protect itself from this clear and present danger. Of all sites in
the body, the endothelium and vascular smooth muscle cells may be at greatest risk of
exposure to heme. Heme greatly potentiates endothelial and smooth muscle cells
damage mediated by leukocytes and other sources of reactive oxygen. Heme also
promotes the conversion of low-density lipoprotein and atherosclerotic plaque lipid to
cytotoxic oxidized products.Reb blood cell lysis and oxidation of ferro-hemoglobin during
plaque rupture occur generating ferri- or ferryl-hemoglobin, followed by transfer ofheme
to cells and lipid domain of the arteries representing a severe heme-stress. Moreover in
the process of hemoglobin oxidation protein radicals are also formed.Oxidized
hemoglobin exhibits pro-inflammatory properties. As a defense against such stress,
endothelial and smooth muscle cells upregulate heme oxygenase-1 and ferritin. Heme
oxygenase opens the porphyrin ring, producing biliverdin, carbon monoxide, and a most
dangerous product-redox active iron. The latter can be effectively controlled by ferritin
via sequestration and ferroxidase activity. These homeostatic adjustments have been
shown to be effective in the pathology of endothelium and smooth muscle cells against
the damaging effects of heme and oxidants.
THE POLISH IRON CLUB
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Receptor systems for hemoglobin and heme – role in inflammation
1,2
1
1,2
Pia Svendsen , Anders Etzerodt and Søren K. Moestrup
1
Department of Biomedicine, Aarhus University, Denmark
Inflammation Research, Institute of Molecular Medicine, University of Southern
Denmark
2
The haptoglobin-CD163-heme oxygenase-1 (HO-1) and the hemopexin-CD91HO pathways are efficient captor-receptor-enzyme systems that circumvent hemoglobin
(Hb)/heme-induced toxicity during physiological and pathological hemoglobin/heme
release. The receptors CD163 and CD91 that only display a high affinity for haptoglobin
or hemopexin when these form complex with hemoglobin or heme, respectively, are
expressed in restricted but overlapping subsets of cells. CD163 expressing is largely
restricted to the various type ‘alternatively’ activated ‘M2-like’ macrophage whereas
CD91, besides being highly expressed in the same group of macrophages, also is
expressed in many other cell types like hepacytes, neurones, fibroblast and
syncytotrophoblasts. Removal of hemoglobin and heme and generation of the heme
metabolites indicate an anti-inflammatory role of the pathways. In accordance with this
function the haptoglobin-CD163-HO-1 pathway proteins are regulated by interleukin-6
(IL-6) that is known as an acute phase mediator that lead to a counteracting antiinflammatory response on acute inflammation We have now further explored the antiinflammatory properties of the CD163 by studying the effect of targeting-disruption of
CD163 in inflammatory models.
THE POLISH IRON CLUB
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Regulation and dysregulation of iron and heme metabolism
Francesca Vinchi
Molecular Medicine Partnership Unit,University of Heidelberg & EMBL, Heidelberg,
Germany;
Molecular Biotechnology Center, University of Turin, Turin, Italy
Despite their essential role for fundamental metabolic processes in cells and
organisms, heme and iron, when in excess,could be potentially toxic due to their ability
to participate in ‘Fenton-type’ redox chemistry, thus leading to the generation of highly
reactive hydroxyl radicals that damage lipids, proteins and nucleic acids. Indeed, heme
and iron metabolism needs to be finely regulated.Different tissues and cell types play a
crucial role in heme and iron metabolism: the erythroid compartment that uses most of
the absorbed and recycled iron and synthesizes the highest amount of heme, the
duodenal enterocytes that are responsible for heme and iron absorption into the
circulation, the reticulo-endothelial macrophages that recycle heme-derived iron to
render it available for erythropoiesis and the liver, that serves as the main iron storage
and heme-detoxifying tissue.
The intracellular amount of both heme and iron is controlled at multiple levels: uptake,
utilization, storage and export in the case of iron; synthesis, incorporation
intohemoproteins, degradation and trafficking in that of heme. Additionally extracellular
heme and iron scavenging by Hemopexin and Transferrin (Tf), respectively, regulates
cell heme and iron uptake and prevents tissue oxidation.
Disruptions in iron and heme homeostasis, from both deficiency and overload, account
for some of the most common human diseases.
Iron metabolism is balanced by two coordinated regulatory systems, one that functions
systemically to maintain physiological Tf saturation and relies on the hormone hepcidin
(Hamp) that controls systemic iron influx via the iron exporter ferroportin (FPN), and
another that predominantly controls cellular iron metabolism through iron-regulatory
proteins (IRPs) that bind iron-responsive elements (IREs) in regulated mRNAs. Inherited
and acquired disorders that perturb hepcidin production, consequently cause iron
deficiency (high hepcidin levels) or iron overload (hepcidin deficiency).
Similarly heme metabolism is balanced by different mechanisms, one that works
systemically by scavenging free hemoglobin and hemein order to avoid heme-mediated
tissue oxidative damage, and a second one that maintains intracellular heme at a
physiological concentration by coordinating its rate of synthesis, degradation and export.
A misbalance in these systems leads to tissue heme overload (sickle cell anemia,
thalassemia, malaria, HO1 deficiency) or heme deficiency (porphyria, sideroblastic
anemia).
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Mechanisms of heme iron absorption in young pigs orally supplemented with
hemoglobin
1
1
1
1
2
Robert Staroń , Paweł Lipiński , Anna Gajowiak , Ewa Smuda , Tomasz Dziaman ,
3
3
4
Wojciech Krzeptowski , Małgorzata Lenartowicz , Małgorzata Gajewska , Marek
5
5
6
6
Pieszka , Dorota Bederska-Łojewska , Dorine Swinkels , Rachel van Swelm , Rafał
1
StarzyńskI
1
Department of Molecular Biology , Institute of Genetics and Animal Breeding PAS,
Poland
2
Depatment of Clinical Biochemistry, Collegium Medicum UMK, Poland
3
Department of Genetics and Evolution, Jagiellonian University, Poland
4
Departament of Physiological Sciences, Warsaw University of Life Sciences, Poland
5
Department of Animal Nutrition and Feed Science, National Research Institute of
Animal Production, Poland
6
Department of Laboratory Medicine, Radboud University Medical Centre, Netherlands
Neonatal iron deficiency anemia (IDA) commonly occurs in young pigs, as a
consequence of a negative iron balance resulting from both insufficient iron stores and
dietary intake. Therefore, to counteract the reduction of iron supply for erythropoiesis
and the development of anemia in piglets, an exogenous iron must be administered.
Heme is an efficient source of iron in the diet, which strongly implies the existence of a
regulated pathway for the absorption of heme-derived iron. However, mechanisms
responsible for heme absorption remain uncharacterized. We aimed at determining the
efficacy of oral heme-iron supplementation of piglets and investigating the molecular
basis of heme-iron absorption. Polish Landrace piglets from three groups were treated
as follows: A. intramuscular injection of iron dextran (FeDex) (150 and 40mg Fe/kg b.w.
on day 3 and 21 after birth, n=8); B. oral supplementation with bovine hemoglobin
(38g/kg diet, 612mg Fe/kg diet, n=15); C. no iron supplementation (n=15). Blood
samples were collected on days 3, 14, 21, 28 after birth. On day 28 piglets were
euthanized and duodenum and liver samples were collected for the expression analysis
of iron-related genes and biochemical iron parameters measurements.
Hematological indices revealed that oral supplementation of piglets with heme
rescues these animals from IDA observed in non-supplemented controls. However, in
contrast to the massive iron deposition observed in the livers of FeDex-treated piglets,
animals supplemented with hemoglobin showed moderate hepatic iron content
suggesting a shift of the heme-derived iron for erythropoiesis. In the duodenum of those
piglets, the expression of apical, basolateral heme (HRG1, HCP1) and non-heme (Fpn)
iron transporters was increased. Interestingly, we found that unlike FeDex-injected
piglets, hemoglobin supplemented animals show very low hepatic expression of
hepcidin mRNA as well as serum and urine hepcidin levels (comparable to anemic
controls). In conclusion, our results show the effectiveness of oral heme
supplementation in fighting IDA and elucidate duodenal pathways of heme-derived iron
absorption and their regulation.
Supported by NCN grant no. 2012/05/E/NZ5/02126.
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Identification of reticuloendothelial cells in kidneys of young mice and their
plausible function in iron metabolism
1
2
1
Aleksandra Bednarz , Paweł Lipiński , Małgorzata Lenartowicz
1
Department of Genetics and Evolution, Institute of Zoology, Faculty of Biology and
Earth Sciences, Jagiellonian University,
2
Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish
Academy of Sciences
Macrophages of reticuloendothelial (phagocytic) system play a crucial role in iron
recycling in the organism due to the ability to recover and recirculate iron from old and
damaged cells, especially from senescent erythrocytes. Resident macrophages are
found in spleen (red pulp macrophages), liver (Browicz-Kupffer cells), lungs (alveolar
cells), central nervous system (microglia), bones (osteoclasts) and bone marrow.
However, very little is known about reticuloendothelial cells present in kidneys under
physiological conditions. In our study, we attempted to identify macrophages in kidneys
of healthy mice during ontogenic development from day 14 after birth until the age of 6
months. Eosin/hematoxylin staining of renal sections from 14-day-old mice revealed a
population of numerous cells showing morphology very similar to that of Browicz-Kupffer
cells. Interestingly, in kidneys of 6-month-old animals these cells were absent. To
confirm the macrophage identity of these cells in kidneys of 14-day old mice, we
performed immunofluorescence (IF) studies using an antibody directed against the
plasma membrane macrophage marker - F4/80 and showed its strong expression in
examined cells. Dual staining with antibodies specific for ferroportin (Fpn) and/or heme
oxygenase 1 (HO1) and F4/80, indicated a strong co-localization of examined proteins
under the confocal microscope, suggesting that both Fpn and HO1 are localized in
kidney macrophages of suckling mice. HO1/F4/80 and Fpn/F4/80 dual staining in
kidneys from 21-, 28-, and 36-day-old mice revealed that macrophages slowly
disappear with age. We hypothesize that kidney macrophages are engaged in iron
recycling from apoptotic cells derived from the reconstruction of kidney during the
process of organogenesis, which is accomplished in 13-day old mice. Macrophages
digest apoptotic cells in the kidney, recover and recycle their iron in a HO1- and Fpndependent process. Iron metabolism in young mice must be very strictly regulated
because their molecular mechanism of iron absorption are not fully developed. Under
such conditions recovering and recirculation of iron by kidney macrophages may be an
important element of systemic iron management.
Supported by NCN grant no. 2012/05/B/NZ4/02423.
THE POLISH IRON CLUB
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Cobalt protoporphyrin: unexpected effect on mobilization of hematopoietic cells
1
1
1
1,2
1
1
A. Szade , K. Szade , W. N. Nowak , K. Bukowska-Strakova , J. Dulak , A. Józkowicz
1
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology,
2
Department of Clinical Immunology, Polish-American Institute of Pediatrics,
Jagiellonian University Medical College, Krakow
Heme oxygenase-1 (HO-1) is an enzyme degrading heme into ferrous ions, carbon
monoxide and biliverdin, which is subsequently reduced to bilirubin. Cobalt
protoporphyrin IX (CoPP) is an inducer of HO-1 expression and activity. We have
previously observed that treatment of mice with CoPP increases the number of
leukocytes in the blood. The aim of the study was to evaluate the effect of CoPP on
hematopoietic cell populations.
Mice were injected intraperitoneally with CoPP (10 mg/kg) or G-CSF (250 µg/kg)
once a day, for 5 days. Six hours after the last dose mice were sacrificed, and both
peripheral blood and bone marrow samples were collected. Mice treated with CoPP and
GCSF had increased number of leukocytes in the blood compared to control mice.
Relative spleen weight was similarly increased after CoPP and G-CSF treatments.
The populations of leukocytes, which were affected by CoPP administration, were
characterized in more detail using a flow cytometer. Treatment with CoPP resulted in
+
+
increased number of circulating granulocytes (CD11b Ly6C Ly6G ) in the blood.
However the granulocytes mobilized by CoPP were more mature than granulocytes
mobilized by GCSF, what was demonstrated by higher granularity and Ly6G expression.
+
+
CoPP induced mobilization of KLS (c-Kit Lin Sca-1 ) cells to the blood. We observed
+
increased numbers of KLS CD48 CD150 , KLS CD48+CD150+CD34+ and multipotent
progenitors (KLS CD48+CD150-CD34+) in the blood of mice treaded with CoPP.
Numbers of committed progenitors (KLS-, c-Kit+Lin-Sca-1-): granulocyte-macrophage
progenitor cells (GMP, KLS- CD48+CD150-CD34+) and megakaryocyte–erythroid
progenitor cells (MEP, KLS- CD48+CD150+CD34-) were also increased in the blood in
response to CoPP treatment.
To conclude, administration of CoPP results in the mobilization of granulocytes and
hematopoetic progenitor cells into the blood.
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Genetic background influences iron homeostasis and hepatic injury in chronic
hepatitis C patients
1
1
1
1
1
2
Woziwodzka , A. Bernat , M. Rybicka , A. Wróblewska , K.P. Bielawski , K. Sikorska
2
Medical University of Gdansk, Department of Tropical Medicine & Epidemiology,
Gdańsk, Poland
1
Intercollegiate Faculty of Biotechnology UG-MUG, Laboratory of Molecular Diagnostics,
Gdańsk, Poland
Iron, involved in several crucial metabolic pathways, can serve as both an
essential nutrient and a potential toxin. Clinical manifestation of iron overload, observed
in patients with hereditary hemochromatosis, includes iron storage in the liver, often
leading to progressive fibrosis, cirrhosis, and eventually hepatocellular carcinoma. It is
supposed that even modest symptoms of iron overload may increase the severity and
progression of other liver diseases. Mild to moderate iron overload is common among
patients with chronic hepatitis C (CHC). Excessive iron accumulation in the liver is
hepatotoxic and thus may lead to poor clinical outcome in CHC patients. The most
common genetic factor leading to iron overload is polymorphism in HFE gene, encoding
protein that regulates iron absorption from the gastrointestinal tract. Three
polymorphisms in HFE gene have been described: C282Y, which is associated with iron
overload symptoms and is present in 85-90% of patients with hemochromatosis, and
H63D and S65C, for both of which clinical significance for iron overload is still unclear.
Apart from HFE gene, polymorphisms in TF gene encoding transferrin and TFR2 gene
encoding transferrin receptor protein 2, have been demonstrated to affect iron
homeostasis.
The aim of the study was to assess the impact of polymorphisms in HFE and
other genes (TF, TFR2, HAMP, HDAC, CYBRD1) relevant for iron homeostasis, on both
serum iron parameters and histopathological markers of liver injury in patients with
CHC. 250 individuals with confirmed CHC were enrolled in the study. Polymorphisms in
selected genes were determined with MALDI-TOF mass spectrometry using Sequenom
MassARRAY platform, and correlated with ALT activity, serum iron overload parameters
(serum iron, transferrin saturation, serum ferritin) as well as histopathological
parameters of liver injury and iron overload (inflammation, fibrosis and iron deposits in
liver tissue).
Patients carrying HFE C282Y polymorphism more often developed iron deposits
in the liver. Elevated parameters of serum iron overload were observed in CYBRD1
variant patients. Interestingly, the 0.46 frequency of C allele in TFR2 gene (rs7385804)
observed in this study was significantly higher than its 0.31 frequency in a general
European population, suggesting its role as a potential risk factor in course of HCVrelated disease. To conclude, this study provided evidence that genetic background
might influence iron homeostasis in CHC patients.
THE POLISH IRON CLUB
18
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Il-28b polymorphisms and iron homeostasis disturbances in relation to hepcidin
expression in chronic hepatitis c.
1
1
1
2
1
A. Wróblewska , A. Bernat , A. Woziwodzka , J. Markiewicz , K.P. Bielawski , T.
2
3
Smiatacz ,K. Sikorska
1
Intercollegiate Faculty of Biotechnology UG-MUG, Laboratory of Molecular Diagnostics,
Gdansk, Poland
2
Department of Infectious Diseases, Medical University of Gdansk, Poland
3
Department of Tropical Medicine and Epidemiology, Medical University of Gdansk,
Poland
Hepatitis C virus (HCV) infection pose an important healthcare problem
worldwide due to a poor response to common therapies and a prevalent development of
chronic hepatitis C (CHC). Iron overload, frequently observed in CHC, is an important
predictive marker for treatment failure and progression of liver fibrosis.Three major
SNPs in the region of IL-28B gene, encoding interferon λ3: rs12979860, rs8099917 and
rs12980275were identified as strong predictors of sustained viral response in HCVinfected patients.Expression of hepcidin(HAMP) gene encoding iron-regulatory hormone
is regulated by inflammatory cytokines and plasma ironand could be a link between
systemic iron status and immune response.
In 192 CHC patients 3 IL-28B SNPs: rs12979860, rs8099917 and rs12980275
were genotyped using AS-PCR. HAMP gene expression in 185liver biopsies was
determined using real time PCR in relation to GUS. In recruited patientsbiochemical
analysisof liver function tests, serum iron and ferritin concentrations and transferrin
saturation, and assessment of inflammation, fibrosis, steatosis, hepatocyte iron deposits
inliver biopsy specimens were performed.
Unfavourable IL-28B genotypes rs12979860 TT and rs8099917 GG were
associated with higher incidence of systemic iron overload (OR 2.8, P=0.004 and OR
2.9, P=0.03, respectively).Patients possessing either of 3 unfavourable IL-28B
genotypes had a significantly elevated serum iron levels.The presence of favourable
haplotype rs12979860 C, rs8099917 T and rs12980275 A decreased chances for
systemic iron overload (OR 0.3, P=0.002). The presence of unfavourable genotype
rs12979860 TT or rs8099917 GG predisposes to more intense steatosis (P values 0.03
and 0.008, respectively) and favourable genotype rs12979860 CC to an increased
degree of inflammation in liver tissue (P=0.03).
Downregulated expression of the HAMP gene correlated with the lack of serum
markers of iron overload as well as the presence of favourable rs12979860 CC,
rs8099917 TTor rs12980275 AA genotype (P values 0.001, 0.003, 0.3 and 0.005,
respectively).
Conclusion: Our study points out the existence of an important link between
systemic iron overload in CHC patients and genetic background of immune response to
HCV infection. Iron overload observed frequently in CHC may be associated with an
aberrant immune response to HCV infection.
THE POLISH IRON CLUB
19
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
The impact of treatment for acute ischemic stroke on hepcidin levels
1
2
1
Artur Słomka , Milena Świtońska and Ewa Żekanowska
1
Department of Pathophysiology, Nicolaus Copernicus University in Toruń, Ludwik
Rydygier Collegium Medicum in Bydgoszcz, Poland
2
Neurology and Stroke Care Unit, Jan Biziel University Hospital № 2, Bydgoszcz,
Poland
A series of studies published over 15 years ago have shown that hepcidin and
soluble hemojuvelin (sHJV) play a fundamental role in iron homeostasis. Nevertheless,
iron metabolism in ischemic stroke remains poorly characterized. Therefore, in this
study, we examined iron homeostasis proteins in relation to type of treatment and time
of sampling in patients with acute ischemic stroke.
Subjects included 31 patients diagnosed with acute ischemic stroke. Whole blood
st
th
was collected twice: at the time of diagnosis (1 day) and one week after (7 day), and
after centrifugation plasma samples were stored frozen. Patients were grouped
according to type of treatment: group I – patients receiving intravenous alteplase
®
(recombinant tissue plasminogen activator, Actilyse 50 ) at 0.9 mg/kg within 4½ hours
of the stroke onset (n=12) and group II – patients not treated with thrombolysis (n=19).
®
Of the 19 patients in group two, 11 patients received enoxaparin sodium (Clexane ) with
the usual pharmacologic doses given by subcutaneous injection. ELISA kits were used
to determine hepcidin (25–amino acid form), sHJV and soluble transferrin receptor
st
th
(sTfR) levels on the 1 and on the 7 day.
We first analyzed hepcidin, sHJV and sTfR levels on the 1st day and on the 7th
day after stroke diagnosis. It was found that the levels of hepcidin, sHJV and sTfR on
the 1st day were similar to the levels on the 7th day (median, 19.82 ng/mL vs 17.35
ng/mL; 443.80 ng/mL vs 386.27 ng/mL; 0.78 µg/mL vs 0.67 µg/mL, respectively). Next,
we sought to examine whether stroke therapy has an impact on the levels of iron
homeostasis proteins. We failed to find a difference in iron homeostasis proteins levels
st
between the two treatment groups on the 1 day. In contrast, significantly lower hepcidin
th
levels were found on the 7 day in the non–thrombolysis group when compared to the
thrombolysis group (median, 16.21 ng/mL vs 22.16 ng/mL; p=0.04).
Acute ischemic stroke patients not treated with rtPA showed lower levels of
hepcidin compared with subjects treated with intravenous rtPA one week after stroke
diagnosis. These findings support that stroke therapy has an influence on plasma
hepcidin levels.
THE POLISH IRON CLUB
20
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Association of serum levels of Fibroblast Growth Factor 23 with iron status,
inflammation andkidney function among patients with early stages of chronic
kidney disease
Ewelina Łukaszyk, Mateusz Łukaszyk, Jolanta Małyszko
2nd Department of Nephrology and Hypertension with Dialysis Unit, Medical University,
M. Sklodowskiej-Curie, Białystok, Poland
In patients with chronic kidney disease (CKD) iron deficiency is frequently
observed. Recent studies have described relationship between iron deficiency,
inflammation, kidney function and Fibroblast Growth Factor 23 (FGF23), however it was
based on patients in end-stage CKD or dialysis population. The aim of this study was to
consider the relation between FGF23 and iron status, inflammation and kidney function
in patients with early stages of CKD.
The analysis included 150 patients. Among patients enrolled in the study, 96
(64%) were in early stage of CKD (2-4) and 47 (31.3%) had functional (FID) or absolute
(AID) iron deficiency. Standard laboratory methods in the hospital central laboratory
were used to measure iron, ferritin, transferrin saturation (TSAT), fibrinogen and
creatinine levels. Serum concentrations of FGF23, high-sensitive C-reactive protein
(hsCRP), growth differentiation factor (GDF-15), hepcidin-25, soluble transferrin
receptor (sTfR), interleukin-6 (IL-6) were assessed with the use of commercially
available assays.
The negative correlation between FGF23 and estimated glomerular filtration rate
(eGFR) was observed (r= - 0.34, P<0.05). FGF23 waspositively correlated with GDF-15
(r=0.22, P<0.05) and IL-6(r=0.16, P<0.05) and negatively with fibrinogen (r=-0.19,
P<0.05). There was no correlation between FGF23 and iron, TSAT, sTfR and ferritin.
Patients with functional iron deficiency had significantly higher concentrations of GDF15, hepcidin-25, ferritin and fibrinogen in comparison with absolute iron deficiency.
FGF23 is not related with classic iron status parameters in patients with early
stages of chronic kidney disease. Functional iron deficiency is related to higher novel
iron status parameters. Impaired renal function is associated with higher serum
concentration of FGF23.
THE POLISH IRON CLUB
21
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Are the mechanisms of nigral cells neurodegeneration in typical and atypical
parkinsonism different - MRI and Mössbauer spectroscopy studies
1
2
3
Radosław Kuliński , Andrzej Friedman , Jolanta Gałązka-Friedman
1
Magnetic Resonance Lab, Bródno Hospital, Warsaw
Department of Neurology, Medical University of Warsaw
3
Faculty of Physics, Warsaw University of Technology
2
Parkinson’s disease (PD) and atypical parkinsonism e.g. progressive
supranuclear palsy (PSP) share some similar clinical features. Their motor symptoms
are due to death of nervous cells in substantia nigra (SN). As the mechanism of the
neurodegeneration in both may be related to the iron mediated oxidative stress injury
and as the role of iron may be eventually assessed in the patients by magnetic
resonance imaging (MRI) and in the post mortem tissue by Mössbauer spectroscopy
(MS), we decided to compare the results of MRI with those obtained with the use of MS
for the concentration of iron.
T1 and T2 relaxation times obtained from SN were determined by MRI from 46
PD patients, 6 PSP and 18 control subjects. MS was performed on 17 PD SN samples,
10 PSP SN samples and 29 controls.
T1 relaxation time from SN was 663±23 ms in PD, 580±12 ms in PSP and 717±4
ms in controls, T2 was 47.2±0.4 ms in PD, 57.3±1.7 ms in PSP and 51.6±0.6 ms in
controls. Iron concentrations as assessed by MS in SN samples were 177±18 ng/mg
wet tissue for PD, 301±26 ng/mg wet tissue for PSP and 177±14 ng/mg wet tissue for
control.
Conclusions: In PSP there is a significant increase of the concentration of iron in
SN compared to control which is not detected in PD. This increase of iron is reflected by
the significant shortening of T1 relaxation time in PSP. This finding is not paralleled by
the change of T2 relaxation time which is the shortest in PD and the longest in control. It
seems therefore that the changes in relaxation times depend not only on the
concentration of iron. Our results suggest that the neurodegeneration in both diseases
may be due to different mechanisms.
THE POLISH IRON CLUB
22
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Overexpression of both non-mutated human SOD1 and mutated SOD1G93A genes
induces changes of iron metabolism genes in mice
Anna Gajowiak, Rafał R. Starzyński, Paweł Lipiński, Robert Staroń, Agnieszka Styś
Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish
Academy of Sciences
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative and paralytic
disease characterized by degeneration and loss of motor neurons in the spinal cord,
brainstem, and motor cortex. Up to 20% of ALS cases are inherited (familial, fALS) and
associated with mutations, usually in the superoxide dismutase 1 (SOD1) gene. Rodent
transgenic models of ALS are frequently used to elucidate a complex pathogenesis of
this disease. Of importance, both ALS patients and animals carrying mutated human
SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation.
The aim of our study was to characterize changes in iron metabolism in transgenic
G93A
mice overexpressing mutant SOD1
gene, a mouse model of ALS. We analyzed
expression of iron-related genes in asymptomatic, 2-month old and symptomatic, 4G93A
month old SOD1
mice. In parallel, respective age-matched mice carrying normal
SOD1 transgene and control mice were analyzed. We demonstrate that the
overexpression of both SOD1 and SOD1G93A genes accounts for a substantial
increase in SOD1 protein levels and activity in selected tissues and that not all the
changes in iron metabolism genes expression are specific for the overexpression of the
mutated form of SOD1.
Supported by NCN grant no. 2011/01/BNZ23/00632
THE POLISH IRON CLUB
23
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Key role of copper in spermatogenesis
Mateusz Ogórek, Paweł Grzmil, Olga Pierzchała, Aleksandra Bednarz, Małgorzata
Lenartowicz
1
Department of Genetics and Evolution, Institute of Zoology, Faculty of Biology and
Earth Sciences, Jagiellonian University, Kraków, Poland
Copper is a trace element essential for the functioning and survival of every cell.
Though systemic and cellular copper metabolism is widely studied, little attention is paid
to the role of copper in the male reproductive system. As shown in a murine model of
Menkes disease, this disorder of copper metabolism leads to morphological changes in
testis, cell damage, decrease in sperm quality and changes in hormone levels. All these
changes found in mutant mice are caused by the toxic effects of high copper level in the
testis, which results from the dysfunction of copper transporter Cu-transporting ATPase,
ATP7A. On the other hand, copper, which provides a signal to begin the process of
meiosis during spermatogenesis, is necessary in the male germ cells production,. All
this data indicate that copper plays an important role in the production of sperm.Toxicity
and indispensability of this trace element requires therefore a strict regulation of its
concentration at the cellular level. The concentration of copper, changing with the
successive stages of spermatogenesis, leads to a change of the whole suit of protective
proteins such as SOD1 or HSP90. We found that ATP7A is expressed in the testis but
its expression is restricted to Leydig cells, Sertoli cells and primary spermatocytes.
Furthermore, we found that in Menkes disease mice copper level in the testis is
elevated. Results of our studies indicate for the specific compensation mechanism when
copper concentration increases in testis. It depends on the induction of ceruloplasmin
expression, which binds free copper ions inside the cell.
Supported by NCN grant no. 2012/05/B/NZ4/02423.
THE POLISH IRON CLUB
24
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
The influence of iron availability on the cell viability and the expression of genes
associated with intracellular iron metabolism within rat H9C2 cardiomyocytes and
L6G8C5 myocytes cultured either in static conditions or upon mechanical stretch
1
2
2
3
4
1,4
M. Kasztura , M. Stugiewicz , K. Kobak , J. Bania , W. Banasiak , P. Ponikowski , EA.
1,4
Jankowska
1
Wroclaw Medical University, Department of Heart Diseases, Wroclaw, Poland
Wroclaw Medical University, Students' Scientific Organisation, Department of Heart
Diseases, Wroclaw, Poland
3
Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland
4
Military Hospital of Wroclaw, Cardiology Department, Wroclaw, Poland
2
Iron is presumed to affect the functioning of cardiomyocytes and skeletal
myocytes. The presence of proteins involved in intracellular iron metabolism in these
cells is anticipated.
H9C2 rat adult cardiomyocytes and L6 rat adult skeletal myocytes were cultured
for 24 hours in static conditions and when exposed to mechanical stretch in optimal or
reduced (iron chelation using deferoxamine) or increased (supplementation of
ammonium ferric citrate) iron concentrations in cultured media. We analysed the mRNA
expression of HIF-1α (hypoxia and iron depletion indicator), ferritin heavy and light
chains (FTH and FTL; iron storage proteins), ferroportin type 1 (FPN1; iron exporter),
transferrin receptor type 1 (TfR1; iron importer), hepcidin (HAMP; iron metabolism
regulator) using qPCR, the level of respective proteins using Western Blot, and the cell
viability using FACS.
In static conditions, cardiomyocytes exposed to reduced iron concentrations in
the medium demonstrated as compared to standard conditions an increased mRNA
expression of HIF-1α (r=0.96, p<0.001) and a decreased mRNA expression of FTH,
FTL, FPN1, HAMP (all r<-0.7, p<0.001). The increased TfR1 expression (r=0.7, p<0.05)
reflected a facilitated iron entrance to the cells. The inverse changes occurred in cells
exposed to increased iron concentrations in the medium. The same pattern of mRNA
expressions was observed in myocytes. There were strong relations between analogous
genes in both cell lines (all r>0.9, p<0.001). The mRNA expression corresponded with
the profile of respective proteins. When exposed to mechanical stretch, both cell lines,
when cultured in reduced iron availability, demonstrated an increased mRNA expression
of TfR1 (both r=0.96, p<0.001) and a reduced mRNA expression of FPN1 (both r= 0.96, p<0.001) similarly as in static conditions. It was accompanied by an increased
HAMP mRNA expression in both cell types (both r>0.6, p<0.01 ), suggesting HAMP to
be involved in the local regulation of iron metabolism. The culture of both cell types in
non-optimal iron supply decreased their viability by 20% in both static conditions and
when exposed to mechanical stretch.
Rat cardiomyocytes and skeletal myocytes contain genes involved in intracellular
iron metabolism and respond similarly to changing iron availability in static conditions.
The application of mechanical stretch modulates responses to changing iron availability
in these cells. Iron depletion and excess impair the viability of the cells.
Supported by NCN grant 2012/05/E/NZ5/00590
THE POLISH IRON CLUB
25
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Influence of hypoxia on the molecular machinery involved in iron metabolism
within rat H9C2 cardiomyocytes and L6G8C5 myocytes cultured in differential iron
availability
1
2
1
3
Magdalena Stugiewicz , Monika Kasztura , Kamil Kobak , Jacek Bania , Waldemar
4
2,4
2,4
Banasiak , Piotr Ponikowski , Ewa A. Jankowska
1
Wroclaw Medical University, Students' Scientific Organisation, Department of Heart
Diseases, Wroclaw, Poland
2
Wroclaw Medical University, Department of Heart Diseases, Wroclaw, Poland
3
Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland
4
Military Hospital, Cardiology Department, Wroclaw, Poland
Hypoxia is expected to affect the functioning of cardiomyocytes and skeletal
myocytes. Hypoxia-induced alteration of the cellular machinery involved in iron
metabolism is anticipated.
H9C2 rat adult cardiomyocytes and L6 rat adult skeletal myocytes were cultured
for 48 hours in hypoxia conditions (1% O2) in optimal or reduced (iron chelation using
deferoxamine, DFO), or increased (supplementation of ammonium ferric citrate, AFC)
iron concentrations in cultured media. We analysed the mRNA expression of HIF-1α
(hypoxia and iron depletion indicator), ferritin heavy and light chains (FTH and FTL; iron
storage proteins), ferroportin type 1 (FPN1; iron exporter), transferrin receptor type 1
(TfR1; iron importer), hepcidin (HAMP; iron metabolism regulator) using qPCR, the level
of respective proteins using Western Blot, and the cell viability by means of MTS.
In hypoxic conditions both cardiomyocytes and myocytes exposed to DFO
treatment demonstrated as compared to standard conditions an increased
mRNA expression of HIF-1α (r=0.93, p<0.01), indicating low oxygen status and depleted
intracellular iron. It was accompanied by an increased HAMP mRNA expression in both
cell types (both r>0.9, p<0.01), suggesting HAMP to be involved in the local regulation
of iron metabolism. The increased TfR1 expression (r=0.93, p<0.01) in both cell types
reflected a facilitated iron entrance to the cells. Unlike normoxia conditions, the DFO
treatment upon low oxygen status caused an increase in mRNA expression of FTH and
FTL (both r>0.9, p<0.01) in L6 cells suggesting cellular response to stress condtions,
and an increased FPN1 expression (r=0.93, p<0.01). There was strong relation between
HIF-1α and HAMP genes in each cell line (both r>0.9, p<0.01). Both cell lines exposed
to AFC treatment demonstrated a decreased mRNA expression of TfR1 (r=-0.9, p<0.01)
and an increased mRNA expression of FTH, FTL and FPN1 (all r>0.9, p<0.01). Hypoxic
conditions caused the 25% (DFO) or 20% (AFC) decrease in cardiomyocytes viability
and 27% (DFO) or 23% (AFC) decrease in myocytes viability.
In hypoxia conditions both cardiomyocytes and myocytes respond to changing
iron avalaibility. Hypoxia-induced alteration of iron metabolism genes may intervene with
the stress response of myocytes.
Supported by NCN grant no. 2012/05/E/NZ5/00590
THE POLISH IRON CLUB
26
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Still alive: mesenchymal stromal cells are resistant to oxidative stress despite the
low level of heme oxygenases
W. N. Nowak, H. Rostinki, J. Markiewicz, K. Barthenheier, C. Cauvin, N. KachamakovaTrojanowska, J. Dulak and A. Józkowicz
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków, Poland
Mesenchymal stromal cells (MSC) are multipotent cells derived from adult tissues,
able to differentiate in vitro to adipocytes, osteoblasts or chondrocytes. Such cells are
widely studied as promising tools in regenerative medicine. However, success of cellular
therapy depends on the cell survival upon the administration to the patient. Heme
2+
oxygenase-1, enzyme that degrades heme to billiverdin, carbon monoxide and Fe , is
important cytoprotective and antioxidant factor, which can affect stem cell performance.
Therefore, the aim of our study was to characterize murine bone marrow-derived
mesenchymal stromal cells lacking functional Hmox1 gene.
We employed bone marrow-derived mesenchymal stromal cells (BM-MSC)
+/+
-/isolated from Hmox1 or Hmox1 mice. Using flow cytometry we characterized BM+/+
-/and Hmox1 cells were differentiated to adipocytes,
MSC phenotype. Hmox1
osteoblasts and myofibroblasts what was confirmed with analysis of marker expression.
Then, we analyzed the MSC response to the stressors such as H2O2, hemin or
increased glucose concentration.
Both MSC Hmox1+/+ and Hmox1-/- showed similar phenotype, differentiation
capacities and production of cytokines or growth factors. Interestingly, Hmox1+/+ and
-/Hmox1 cells showed similar survival in response to 50 µmol/L hemin even in the
presence of increased glucose concentration, conditions that were unfavorable for
-/+/+
Hmox1 bone marrow-derived proangiogenic cells (BDMC). Surprisingly, Hmox1 and
-/Hmox1 MSC stood firm against up to 100 µmol/L hemin for 6 hours. What is more,
+/+
+/+
MSC but not Hmox1
fibroblasts retained low ROS levels even after
Hmox1
prolonged incubation with 50 µmol/L hemin. However, both cell types have comparable
level of Hmox1 expression and similarly increase its levels in response to hemin.
Concluding, lack of Hmox1 does not influence MSC phenotype and tested
functions. MSC Hmox1-/- showed higher resistance to the hemin treatment than BDMC.
MSC Hmox1+/+ better withstand hemin than fibroblasts regardless of the similar Hmox1
expression level. The mechanism of such resistance must be further evaluated.
THE POLISH IRON CLUB
27
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Binding of Hsp70 chaperone Ssq1, and frataxin homologue Yfh1, to the iron-sulfur
cluster scaffold Isu1 protein is mutually exclusive
1
1
2
2
Mateusz Manicki , Julia Majewska , Szymon Ciesielski , Brenda Schilke , Anna
1
1
2
1,2
Blenska , Jacek Kominek , Elizabeth A. Craig , Jarosław Marszałek , Rafał
1
Dutkiewicz
1
Faculty of Biotechnology, University of Gdansk, Poland
Department of Biochemistry, University of Wisconsin-Madison, Madison, USA
2
Fe-S clusters are prosthetic groups critical for the activity of many proteins. In the
mitochondria of yeast Saccharomyces cerevisiae, Fe-S cluster biogenesis is an
essential, complex process involving a dynamic interplay of protein:protein interactions
centered on the 14 kDa protein Isu. Isu serves as a scaffold on which Fe-S clusters are
assembled in a process involving the formation of an "assembly complex" composed of
Isu, Nfs1-Isd11 (the cysteine desulfurase that serves as a sulfur donor) and Yfh1 (the
yeast frataxin homolog, a regulator of Nfs1-Isd11 activity and/or iron donor). Nfs1-Isd11
and Yfh1 interact with each other and with Isu. The molecular chaperones Jac1 and
Ssq1, the J-protein/Hsp70 pair, are required for transfer of Fe-S clusters from Isu to
recipient apo-proteins. Little is known however, about the molecular basis of the
transition from the assembly complex to the transfer reaction. Our long-term goal is to
dissect the interactions between the assembly complex and Jac1/Ssq1 to understand
how these chaperones drive this transition.
Here, we report that by using available structural and biochemical data we have
modeled the structure of the assembly complex and predicted surface exposed residues
critical for individual protein:protein interactions (Nfs1-Isd11:Yfh1 and Isu:Yfh1). We
verified our predictions biochemically using a semi-quantitative in vitro pull down assay
utilizing Isu-GST and Yfh1-GST fusion proteins. We observed that the reconstituted
Isu:Yfh1:Nfs1-Isd11 assembly complex is stable only if both Yfh1:Nfs1-Isd11 and
Yfh1:Isu interactions were intact. Disruption of either of these protein:protein
interactions, by substituting key amino acid residues, resulted in dissociation of Yfh1
from the assembly complex. Moreover, we observed that Isu:Yfh1 interaction involves a
PVK sequence motif of Isu, which is also the site of interaction between Isu and Hsp70,
Ssq1. Thus, interactions between Isu and either Yfh1 or Ssq1 are likely mutually
exclusive. We hypothesis, that this mutually exclusive binding could have functional
consequences for the transition from the assembly complex to the transfer reaction.
THE POLISH IRON CLUB
28
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Homocysteine impairs iron metabolism in HUVEC cells
1,2
1
1
Jędrzej Antosiewicz , Andżelika Borkowska , Katarzyna Kaczor
1
Department of Bioenergetics and Physiology of Exercise, Medical University of
Gdansk, Poland
2
Department of Biochemistry University School of Physical Education and Sport
Gdansk
Homocysteine (Hc) is considered to be a risk factor for arteria dysfunction and
vasculopathic effects of homocysteine are iron-dependent. For example iron sucrose
exacerbates and iron chelation alleviates homocysteine-induced reduction in flowmediated dialation. However, the impact of homocysteine on iron metabolism is not well
understood.
To explore the effects of Hc on iron metabolism, we used human umbilical vein
endothelial cells (HUVEC) as a model. Ferritin concentration, transferrin receptor 1 and
ferroportin levels were measured. Hc treatment induced changes in both ferritin L and H
level. In particular, a minor decrease of both ferritins were observed during the first two
hours of Hc treatment, whereas the substantial rises were observed afterwards. Some
equivocal changes in ferroportin level were also noticed which indicates that Hc may
modify cells’ ability to export iron. In addition, the effects of Hc on serine/threonine
kinase (Akt) signaling pathway were measured. We observed that the down regulation
of Akt signaling induced by Hc or by transfection of the cells with siRNA against Akt1,
Akt2 or Akt3 led to increase in both, ferritin L and H proteins level. Altogether, these
data show that Hc induces changes in HUVEC iron metabolism and it is, at least
partially, mediated by changes in Akt signaling pathway. Our observations are in
agreement with previous studies where the increase of iron accumulation was noticed in
people with insulin resistance.
THE POLISH IRON CLUB
29
INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Impact of the copper deficiency on the expression of the gene Ctr1 and cellular
localization of CTR1 protein in mice with mutation in the Atp7a gene.
Olga Pierzchała, Aleksandra Bednarz, Małgorzata Lenartowicz
Department of Genetics and Evolution, Institute of Zoology, Faculty of Biology and Earth
Sciences, Jagiellonian University, Kraków, Poland
Copper is an essential trace element which takes part in multiple cellular
processes including mitochondrial oxidative phosphorylation, neurotransmitter synthesis
and maturation, free-radical detoxification, iron metabolism and pigmentation. In
mammals cellular copper import is primarily mediated by protein called CTR1. This
protein belongs to the family SLC31 of copper transporters and has a very high affinity
+
to Cu ions This Cu-specific carrier has been identified in several tissues including
intestine, liver, kidneys, brain and mammary gland. In the cells CTR1 is predominantly
found at the plasma membrane and constitutively cycles between plasma membrane
and intracellular vesicles. Its location may be modified by a few factors including cell
type and extracellular copper concentration. It is known that the cell surface pool of
CTR1, in response to elevated copper concentration, is rapidly internalized and
degradated.
Mutation in X-linked ATP7A/Atp7a gene, encoding a Cu-transporting ATPase ATP7A protein, causes Menkes disease in humans and mottled mutation in mice. In
both Menkes patients and mutant mice defective intestinal absorption leads to copper
deficiency in various organs with subsequent kidney and small intestine Cu
accumulation. In our research we analyzed the expression of the Ctr1 gene using RealTime PCR in kidneys derived from the mutant mice. We also investigated CTR1 cellular
localization in the mouse kidneys and we found that elevated copper concentration in
the mutant cells was correlated with the increase in Ctr1 gene expression and changed
intracellular localization of the CTR1 protein.
Supported by NCN grant no. 2012/05/B/NZ4/02423.
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Is iron chelation important in preventing glycation of bovine serum albumin in
vitro?
1
1,2
1
Sabina Galiniak , Grzegorz Bartosz , Izabela Sadowska-Bartosz
1
Department of Biochemistry and Cell Biology, University of Rzeszów, Poland
Department of Molecular Biophysics, University of Łódź, Poland
2
It has been proposed that decompartmentalized metal ions are a major
contributor to the development of diabetic complications and that metal chelation may
be responsible for the inhibition of advanced glycation end products (AGE) formation by
a variety of drugs commonly used for the treatment of diabetic complications. However,
when studying the glycation of bovine serum albumin (BSA) by sugars, glyoxal (GO)
and methylglyoxal (MGO) we found limited or no protection by metal chelators such as
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DETAPA)
or nitrilotriacetic acid (NTA) in the simple in vitro system. This prompted us to perform a
systematic comparison of the chelating activity of compounds affecting the rate of BSA
glycation. We compared the iron-chelating activity (estimated by the ferrozine test) and
the extent of inhibition of BSA glycation in vitro induced by glucose, fructose and ribose
of 38 compounds and found no correlation betweeen these parameters. In line with
these results, treatment of the glycating solution with the metal ion-binding resin Chelex
X-100 did not affect the course of glycation. These data indicate that the glycation of
BSA in vitro is iron-independent and that is not a proper system to study the role of
metals in protein glycation.
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Modification of the deoxyribose test to detect strong iron chelators
1
1,2
1
Sabina Galiniak , Grzegorz Bartosz , Izabela Sadowska-Bartosz
1
Department of Biochemistry and Cell Biology, University of Rzeszów, Poland
Department of Molecular Biophysics, University of Łódź, Poland
2
Since introduction, the deoxyribose test based on inhibition of damage to
deoxyribose induced by a Fenton system of hydrogen peroxide, ferric chloride and
ascorbic acid has been frequently used to estimate the reactivity of various compounds
for the hydroxyl radical. The generation of hydroxyl radical in this system is dependent
3+
upon redox cycling via reduction of Fe by ascorbate. We observed that even in the
2+
absence of ascorbate and H2O2, Fe induced oxidative damage to deoxyribose albeit of
lower magnitude. It is not easy to determine whether inhibition of deoxyribose damage
is due to competitive scavenging of the hydroxyl radical or to prevention of the hydroxyl
2+
radical formation by iron chelation strong enough to prevent the participation of Fe in
2+
the Fenton reaction. We studied the effect of chelator concentration at constant Fe
2+
concentration and the effect of Fe concentration at constant chelator concentration on
2+
the deoxyribose damage. The titration of chelators with increasing Fe concentrations
from substoichiometric to suprastoichiometric yielded two types of plots, the
dependence being hyperbolic for most substances but sigmoidal for some chelators.
The same type of dependence was observed in the absence and in the presence of
ascorbate and H2O2. We interpret the sigmoidal dependence of the deoxyribose
damage on Fe2+ concentration, at a fixed inhibitor concentration, with inflection point
2+
corresponding to a 1:1 ratio, as indication of strong Fe binding by a chelator.
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
Why do results of in vivo and in vitro experiments on ferritin iron absorption
greatly differ?
Magdalena Zielińska-Dawidziak
Department of Food Biochemistry and Analysis, Poznań University of Life Sciences,
Poland
Ferritin is a very conservative protein which is present in cells of almost all living
organisms. It has many unique features of which the most crucial is the possibility to
accumulate plenty of iron ions - up to 4500 of atoms in one molecule. Moreover, iron in
mineral core is isolated from the surrounding environment by the protein’s shell. The
protein is thermally stable and resistant to a wide range of pH changes. Ferritin
expression may be increased in plants, both through genetic engineering methods and
modification of plants growth conditions. It is not surprising that the protein has
interested nutritionists as a potential source of iron supplement for individuals suffering
from its deficiency. The ferritin obtained from legumes sprouts was successfully used in
bioactive food production. Many studies were conducted on its bioavailability, but
discrepancies were observed in the obtained results; specifically results from some in
vitro experiments suggest ferritin instability during digestion in the stomach and differ
from those obtained from animal and clinical studies.
Ferritin iron uptake depends on different mechanisms and involves proteins
different from the ones that participate in heme and Fe(II) ions transport. Its availability
is comparable to pharmaceutical preparations and depends on the organism iron status.
These results were confirmed during in vivo experiments. It may be concluded that a
diet composition may strongly influence the stability of ferritin, preventing protein
denaturation by the acid secreted in the stomach. There is also possibility that ferritin
can be renaturated in the small intestine . Incorrect sample preparations for in vitro
experiments were also proven. That is why, even if results of in vitro analyses are
disturbing, they should not restrain ? the studies on plant ferritin nutritional value, but
should constitute a challenge for the future.
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
LIST OF PIC MEETING PARTICIPANTS
Jędrzej ANTOSIEWICZ
Medical University of Gdansk
ul. M. Skłodowskiej-Curie 3a
80 80-210 Gdańsk, Poland
[email protected]
Alicja JÓZKOWICZ
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
[email protected]
Jozsef BALLA
University of Debrecen
Egyetem tér 1, 4032, Debrecen, Hungary
[email protected]
Monika KASZTURA
Institute of Immunology and Experimental
Therapy,
ul. Rudolfa Weigla 12,
Wroclaw, Poland
[email protected]
Grzegorz BARTOSZ
University of Lodz
ul. Narutowicza 65,
90-131 Łódź, Poland
[email protected]
Kamil KOBAK
Wrocław Medical University
Wybrzeże Ludwika Pasteura 1,
50-367 Wrocław, Poland
Aleksandra BEDNARZ
Jagiellonian University
ul. Gronostajowa 9
30-387 Kraków, Poland
[email protected]
Radosław KULIŃSKI
Medical University of Warsaw
ul. Żwirki i Wigury 61
02-091 Warszawa, Poland
[email protected]
François CANONNE-HERGAUX
CHU Purpan Place du Dr Baylac
BP 3028 31024 TOULOUSE Cedex 3, France
[email protected]
Andrzej FRIEDMAN
Bródno Hospital
ul. Kondratowicza 8
03-242 Warszawa, Poland
[email protected]
Anna GAJOWIAK
Institute of Genetics and Animal Breeding PAS
ul. Postepu 36a
05-552 Magdalenka, Poland
[email protected]
Jolanta GAŁĄZKA-FRIEDMAN
Warsaw University of Technology
ul. Koszykowa 75
00-662 Warszawa, Poland
[email protected]
Sabina GALINIAK
University of Rzeszów
ul. Zelwerowicza 4
35-601 Rzeszów, Poland
[email protected]
Małgorzata LENARTOWICZ
Jagiellonian University
ul. Gronostajowa 9
30-387 Kraków, Poland
[email protected]
Ewelina ŁUKASZYK
Medical University of Bialystok
ul. Jana Kilińskiego 1
15-089 Bialystok, Poland
[email protected]
Paweł LIPIŃSKI
Institute of Genetics and Animal Breeding PAS
Jastrzębiec, ul. Postępu 36a
05-552 Magdalenka, Poland
[email protected]
Jolanta MAŁYSZKO
Medical University of Bialystok
ul. Jana Kilińskiego 1
15-089 Bialystok, Poland
[email protected]
Mateusz MANICKI
University of Gdansk, Poland
Jana Bażyńskiego 1A,
80-952 Gdańsk, Poland
THE POLISH IRON CLUB
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INSTITUTE OF GENETICS AND ANIMAL BREEDING PAS
[email protected]
Søren MOESTRUP
University of Aarhus
Ole Worms Allé 3
8000 Aarhus C, Denmark
[email protected]
Rafał R. STARZYŃSKI
Institute of Genetics and Animal Breeding PAS
Jastrzębiec, ul. Postepu 36a
05-552 Magdalenka, Poland
[email protected]
Witold NOWAK
Jagiellonian University
Gronostajowa 7
30-387 Krakow, Poland
[email protected]
Magdalena STUGIEWICZ
Wrocław Medical University
Wybrzeże Ludwika Pasteura 1,
50-367 Wrocław, Poland
Agnieszka STYŚ
Institute of Genetics and Animal Breeding PAS
Jastrzebiec, ul. Postepu 36a
05-552 Magdalenka, Poland
[email protected]
Mateusz OGÓREK
Jagiellonian University
ul. Gronostajowa 9
30-387 Kraków, Poland
[email protected]
Natalia PIEKUŚ
Collegium Medicum of Nicolaus Copernicus
University
ul. Jagiellońska 13-15
85-067 Bydgoszcz, Poland
[email protected]
Olga PIERZCHAŁA
Jagiellonian University
ul. Gronostajowa 9
30-387 Kraków, Poland
[email protected]
Agata SZADE
Jagiellonian University
Gronostajowa 7
30-387 Krakow, Poland
[email protected]
Francesca VINCHI
Heidelberg University Medical Center
Neuenheimer Feld 672, 69120 Heidelberg,
Germany
[email protected]
Izabela SADOWSKA-BARTOSZ
University of Rzeszów
ul. Zelwerowicza 4
35-601 Rzeszów, Poland
[email protected]
Anna WOZIWODZKA
Intercollegiate Faculty of Biotechnology,
University of Gdansk and
Medical University of Gdansk
Kładki 24
80-822 Gdańsk, Poland
[email protected]
Katarzyna SIKORSKA
Medical University of Gdansk
ul. M. Skłodowskiej-Curie 3a
80 80-210 Gdańsk, Poland
[email protected]
Magdalena ZIELIŃSKA-DAWIDZIAK
Poznań University of Life Sciences
ul. Mazowiecka 48
60-623 Poznań, Poland
[email protected]
Artur SŁOMKA
Collegium Medicum of Nicolaus Copernicus
University
ul. Jagiellońska 13-15
85-067 Bydgoszcz, Poland
[email protected]
Robert STAROŃ
Institute of Genetics and Animal Breeding PAS
Jastrzębiec, ul. Postepu 36a
05-552 Magdalenka, Poland
[email protected]
THE POLISH IRON CLUB
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