bsc. (hons)iron transport and liver injury in mouse models of hereditary haemochromatosis roheeth d....
TRANSCRIPT
IRON TRANSPORT AND LIVER
INJURY IN MOUSE MODELS OF
HEREDITARY HAEMOCHROMATOSIS
Roheeth D. Delima
BSc. (Hons)
This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia in the School of Medicine and Pharmacology, April 2013.
”Somewhere, something incredible is waiting to be known.”
- Dr. Carl Sagan (American Astronomer, Writer and Scientist, 1934-1996)
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Contents
Declaration .................................................................................................................................... viii
Acknowledgements ......................................................................................................................... ix
Abstract x
Publications and presentations arising from this thesis ................................................................ xiii
List of Figures ............................................................................................................................... xiv
List of Tables ................................................................................................................................. xiv
Abbreviations ................................................................................................................................. xv
Chapter 1..............................................................................................................................................2
Literature review ...................................................................................................................................2
Introduction...........................................................................................................................................3
1.1 Iron in the human body ...................................................................................................3
1.2 Iron absorption................................................................................................................4
1.2.1 Dcytb ............................................................................................................................5
1.2.2 DMT1 ............................................................................................................................5
1.2.3 Haem uptake ................................................................................................................6
1.3 Iron export ......................................................................................................................7
1.3.1 Ferroportin ..........................................................................................................................7
1.4 Plasma iron .....................................................................................................................9
1.4.1 Transferrin-bound iron ..................................................................................................9
1.4.2 Non-transferrin bound iron ........................................................................................ 10
1.4.3 Transferrin-bound iron uptake ................................................................................... 10
1.4.3.1 Transferrin Receptor 1 (TFR1) .......................................................................... 10 1.4.3.2 Transferrin Receptor 2 (TFR2) .......................................................................... 12
1.4.4 Non-transferrin bound iron uptake ............................................................................ 14
1.4.4.1 Zrt- and Irt-like Protein 14 (ZIP14) .................................................................... 15 1.4.4.2 Ferritin uptake ................................................................................................... 16 1.4.4.3 Haem/Haemoglobin uptake ............................................................................... 16
1.5 Cellular iron ................................................................................................................. 17
1.5.1 Haem synthesis ......................................................................................................... 18
1.6 Iron storage ................................................................................................................. 19
1.6.1 Ferritin ....................................................................................................................... 19
1.6.2 Haemosiderin ............................................................................................................ 20
1.7 Systemic Iron homeostasis ......................................................................................... 21
1.7.1 Hepcidin .................................................................................................................... 21
1.7.2 Iron-dependent -hepcidin signalling ......................................................................... 22
1.7.2.1 BMP/SMAD signalling ....................................................................................... 22 1.7.2.2 Haemojuvelin ..................................................................................................... 22 1.7.2.3 HFE/TFR2 ......................................................................................................... 24
1.7.3 Erythropoietic Hepcidin signalling ............................................................................. 25
iv
1.7.4 Inflammatory Hepcidin signalling .............................................................................. 25
1.8 Cellular iron homeostasis ............................................................................................ 27
1.8.1 Transferrin Receptor 2 (TFR2) regulation ................................................................. 27
1.8.2 Zrt- and Irt-like Protein 14 (ZIP14) regulation ........................................................... 27
1.8.3 Iron Regulatory Element (IRE)/Iron Regulatory Protein (IRP) regulation ................. 28
1.8.4 Ferroportin internalisation by hepcidin ...................................................................... 29
1.9 Hereditary haemochromatosis .................................................................................... 29
1.9.1 Hereditary haemochromatosis (HH) type 1 .............................................................. 29
1.9.2 Hereditary haemochromatosis type 2 ...................................................................... 30
1.9.3 Hereditary haemochromatosis type 3 ...................................................................... 31
1.9.4 Hereditary haemochromatosis Type 4 ..................................................................... 32
1.10 Pathogenesis of HH .................................................................................................... 33
1.11 Iron induced liver injury ............................................................................................... 33
1.11.1 Generation of reactive oxygen species ..................................................................... 34
1.11.2 Lipid peroxidation ...................................................................................................... 34
1.11.3 Lysosmal fragility ....................................................................................................... 35
1.11.4 Mitochondrial damage ............................................................................................... 35
1.11.5 DNA damage ............................................................................................................. 35
1.11.6 Oxidative stress ......................................................................................................... 36
1.11.7 Inflammatory cytokines ............................................................................................. 36
1.12 Present study............................................................................................................... 36
Aims .......................................................................................................................................... 37
Hypothesis 1: ....................................................................................................................... 37 Aim 1: ................................................................................................................................... 37 Hypothesis 2: ....................................................................................................................... 38 Aim 2: ................................................................................................................................... 38 Hypothesis 3: ....................................................................................................................... 38 Aim 3: ................................................................................................................................... 38 Hypothesis 4: ....................................................................................................................... 39 Aim 4: ................................................................................................................................... 39
Chapter 2........................................................................................................................................... 40
Materials and Methods ...................................................................................................................... 40
Materials ............................................................................................................................................ 41
2.1.1 Tissue collection ......................................................................................................... 41
2.1.2 Experimental procedures ........................................................................................... 41
2.1.3 Molecular biology ....................................................................................................... 42
2.1.4 Protein extraction and Western blotting ..................................................................... 42
2.1.5 Equipment .................................................................................................................. 43
2.1.5.1 Balances ................................................................................................................ 43
2.1.5.2 Centrifugation ........................................................................................................ 43
2.1.5.2 Imaging system ..................................................................................................... 43
2.1.5.3 Microscope ............................................................................................................ 43
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2.1.5.4 Peristaltic pump ..................................................................................................... 44
2.1.5.5 pH measurement ................................................................................................... 44
2.1.5.6 Pipettes ................................................................................................................. 44
2.1.5.6 Powerpacks ........................................................................................................... 44
2.1.5.7 Radioactivity measurements ................................................................................. 44
2.1.5.8 Spectrophotometry ................................................................................................ 44
2.1.5.9 Real-time PCR ...................................................................................................... 45
2.1.5.10 Thermocycler ......................................................................................................... 45
2.1.5.11 Western blot transfer apparatus ............................................................................ 45
2.1.6 Location of suppliers .................................................................................................. 45
General methods ............................................................................................................................... 46
2.2.1 Animals ...................................................................................................................... 46
2.2.2 ICP-AES .................................................................................................................... 47
2.2.3 Plasma Iron Assay .................................................................................................... 47
2.2.4 Total Iron Binding Capacity (TIBC) ........................................................................... 48
2.2.5 Non-Transferrin Bound Iron (NTBI) Assay ................................................................ 49
2.2.5.1 Preparation of Tris-carbanatocobaltate(III) trihydrate ....................................... 49 2.2.6 RNA extraction .......................................................................................................... 50
2.2.7 DNase treatment of RNA .......................................................................................... 51
2.2.8 RNA quantification..................................................................................................... 51
2.2.9 Gel Electrophoresis ................................................................................................... 52
2.2.10 Reverse transcriptase-Polymerase Chain Reaction (RT-PCR) ................................ 52
2.2.11 Primers ...................................................................................................................... 53
2.2.12 Protein extraction ...................................................................................................... 55
2.2.12.1 Bicinchoninic acid (BCA) protein assay ........................................................... 56 2.2.13 Statistical analysis ..................................................................................................... 56
Chapter 3........................................................................................................................................... 57
Characterisation of mouse models of hereditary haemochromatosis ............................................... 57
3.1 Introduction .......................................................................................................................... 58
3.2 Methods ............................................................................................................................... 59
3.2.1 Animals ....................................................................................................................... 59
3.2.2 Tissue collection ......................................................................................................... 59
3.2.3 Haematology .............................................................................................................. 59
3.2.4 Plasma iron measurement ......................................................................................... 60
3.2.5 Liver function .............................................................................................................. 60
3.2.6 Hepatic metal measurement ...................................................................................... 60
3.2.7 Gene expression ........................................................................................................ 60
3.2.8 p-Smad 1/5/8 expression ........................................................................................... 60
3.2.9 Statistics ..................................................................................................................... 61
3.3 Results ................................................................................................................................. 61
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3.3.1 Haematology .............................................................................................................. 63
3.3.2 Plasma Iron parameters ............................................................................................. 65
3.3.3 Liver function .............................................................................................................. 67
3.3.4 Liver metal content ..................................................................................................... 69
3.3.5 Liver expression of iron regulatory genes .................................................................. 71
3.3.6 Liver expression of SMAD1/5/8 .................................................................................. 73
3.3.7 Liver expression of iron transport genes .................................................................... 75
3.3.8 Duodenal expression of iron transport genes ............................................................ 77
3.4 Discussion ............................................................................................................................ 79
Chapter 4........................................................................................................................................... 85
Non-transferrin-bound iron transport in hereditary haemochromatosis ............................................ 85
4.1 Introduction .......................................................................................................................... 87
4.2 Methods ............................................................................................................................... 88
4.2.1 Animals ....................................................................................................................... 88
4.2.2 NTBI uptake ................................................................................................................ 88
4.2.3 Tissue collection ......................................................................................................... 89
4.2.4 Plasma iron measurement ......................................................................................... 89
4.2.5 Hepatic iron content ................................................................................................... 89
4.2.6 Statistics ..................................................................................................................... 90
4.3 Results ................................................................................................................................. 90
4.3.1 Tissue iron content ..................................................................................................... 90
4.3.2 Plasma iron parameters ............................................................................................. 91
4.3.3 Plasma NTBI clearance .............................................................................................. 93
4.3.4 Tissue NTBI uptake .................................................................................................... 94
4.4 Discussion .......................................................................................................................... 102
Chapter 5......................................................................................................................................... 107
Disruption of HFE and TFR2 causes iron-induced liver injury in mice............................................ 107
5.1 Introduction ........................................................................................................................ 109
5.2 Methods ............................................................................................................................. 110
5.2.1 Animals ..................................................................................................................... 110
5.2.2 Tissue collection ....................................................................................................... 110
5.2.3 Histology ................................................................................................................... 110
5.2.4 Perls' Prussian blue staining .................................................................................... 111
5.2.5 Haemotoylin & Eosin staining ................................................................................... 111
5.2.6 Immunofluorescence ................................................................................................ 111
5.2.7 Biochemical markers of liver injury ........................................................................... 112
5.2.8 Collagen staining ...................................................................................................... 112
5.2.9 Gene expression ...................................................................................................... 113
5.3 Results ............................................................................................................................... 114
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5.3.1 Iron measurements .................................................................................................. 114
5.3.2 Liver histology........................................................................................................... 116
5.3.3 Biochemical markers of liver injury ........................................................................... 119
5.3.4 Collagen deposition .................................................................................................. 121
5.3.5 Hepatic expression of injury-related genes .............................................................. 125
5.4 Discussion .......................................................................................................................... 127
Chapter 6......................................................................................................................................... 131
Inflammation in mouse models of hereditary haemochromatosis ................................................... 131
6.1 Introduction ................................................................................................................ 132
6.2 Methods ............................................................................................................................. 133
6.2.1 Animals ..................................................................................................................... 133
6.2.2 Tissue collection ....................................................................................................... 133
6.2.3 Plasma iron parameters ........................................................................................... 134
6.2.4 Gene expression ...................................................................................................... 134
6.3 Results ............................................................................................................................... 135
6.3.1 Effect of LPS with time on hepcidin expression ....................................................... 135
6.3.2 Hepatic expression of inflammatory genes .............................................................. 136
6.3.3 Plasma iron parameters ........................................................................................... 138
6.3.4 Hepatic expression of iron regulatory genes ............................................................ 140
6.3.5 Hepatic expression of iron transport genes .............................................................. 142
6.4 Discussion .......................................................................................................................... 144
Chapter 7......................................................................................................................................... 149
General discussion .......................................................................................................................... 149
7.1 Future directions ................................................................................................................ 155
7.1.1 The role of HFE and TFR2 and erythropoiesis ........................................................ 155
7.1.2 NTBI Transporters .................................................................................................... 155
7.1.3 Iron-induction of liver injury ...................................................................................... 156
Chapter 8......................................................................................................................................... 157
Bibliography .................................................................................................................................... 157
viii
Declaration
I, Roheeth Delima declare that this thesis is my own account of my research and contains
as its main content work which has not previously been submitted for a degree at any
tertiary education institution.
…................................................
Roheeth Delima
…................................................
Date
ix
Acknowledgements
Just as it takes a village to raise a child, my Ph.D candidature would not have been
possible without the support and assistance of the following people, to whom I am
eternally grateful
To my supervisors; thank you Professor Debbie Trinder, for giving me this great
opportunity and for your unending support. Dr Anita Chua, my heartfelt thanks for the
lunches, guidance, friendship and for “kicking my arse” when required. To Professor John
Olynyk and Dr Janina Tirnitz-Parker, thank you for all your support and for the promise that
there is a life after a Ph.D.
Thank you Dr Jane Allan, for your concern and advice over the time of my candidature,
your willingness to drop your own work to answer my constant questions has made my
time at Fremantle much easier. To all the past and present members of the Medical
Science labs at Fremantle hospital, thank you for making this such a nuturing and friendly
place to work.
Thank you to Fremantle Hospital Medical Research Foundation for your continued support
of my Ph.D (Warren Jones Scholarship) and external projects (grants).
To my friends, who were always willing to provide a much needed distraction and who
kindly refrained from asking how my thesis was going, thank you for all your patience and
support.
Last but not least, thank you to my family. To my sisters, thank you for putting up with me
during those times (years) when I was not always a pleasure to be around. Finally, thank
you to my parents. Thank you for your un-ending support; both financially and emotionally,
for your guidance and constant offers of assistance, this achievement is just as much
yours as it is mine.
x
Abstract Fundamental biochemical activities, such as, oxygen transport, energy production and
cellular proliferation are all dependent on iron-containing proteins. Although iron is
essential, excess is toxic due to its ability to catalyse the production of reactive oxygen
species and damage cellular macromolecules (Chua et al. 2007). Hereditary
haemochromatosis (HH) is an autosomal recessive disorder in which excessive absorption
of dietary iron leads to iron accumulation in the parenchymal tissues. Excessive iron
accumulation is most prominent in the liver as well as in the pancreas, pituitary, heart,
joints and skin and may lead to liver fibrosis, cirrhosis and hepatocellular carcinoma,
diabetes mellitus, impotence, cardiac failure, arthritis and skin hyperpigmentation. There
are five types of HH caused by mutations in genes that encode proteins involved in the
synthesis of hepatic regulatory peptide hepcidin, and its receptor, ferroportin that regulate
iron metabolism.
The general aim of this study was to characterise the roles of the proteins;
haemochromatosis protein (HFE) and transferrin receptor 2 (TFR2) which are mutated in
HH type 1 and 3, respectively, in iron transport and the regulation of iron metabolism.
Disruption of both HFE and TFR2 in mice (Hfe-/-xTfr2mut) resulted in a more severe iron
loaded phenotype with increased plasma iron, non-transferrin bound iron (NTBI)
concentration, transferrin saturation, and liver iron content compared with mice with
disruption in either HFE (Hfe-/-) or TFR2 (Tfr2mut) alone. Hfe-/-xTfr2mut mice had elevated
liver Bmp6 mRNA expression consistent with increased liver iron content. However,
disruption of Hfe and Tfr2 expression resulted in ineffective liver p-Smad 1,5,8 signalling
leading to reduced liver Hamp1 expression. The more severe iron-loaded phenotype in
Hfe-/-xTfr2mut mice compared with single mutant mice suggests a model of iron-dependent
regulation of hepcidin where both HFE and TFR2 act as plasma iron sensors via parallel
and possibly converging signalling pathways.
xi
Decreased hepcidin expression results in excessive dietary iron absorption and iron
release from macrophages, which saturates plasma transferrin and leads to the increased
presence of a toxic from of iron known as NTBI. Measurement of in vivo NTBI transport in
mouse models of HH showed that NTBI was cleared rapidly from the circulation in all
mouse models of HH, with most of the NTBI taken up by the liver and to a lesser degree
by the kidneys, pancreas and heart. Uptake of NTBI was greater in Hfe-/-xTfr2mut mice than
in Hfe-/- and Tfr2mut mice, which in turn had greater uptake than wild-type mice. NTBI
uptake was positively correlated with both plasma NTBI levels and iron content in the liver,
kidney, pancreas and heart suggesting that NTBI uptake contributes to tissue iron
overload in HH.
Free iron can generate reactive oxygen species (ROS) which may cause oxidative tissue
damage. In association with the previously mentioned severe iron-loaded phenotype, Hfe-/-
xTfr2mut mice had elevated plasma alanine transaminase activity, mild hepatic
inflammatory cell infiltration with scattered foci of CD45+ leukocytes co-localised
predominately with ferritin in portal regions of the liver. Elevated hydroxyproline levels, and
Sirius red and Trichrome staining demonstrated marked portal tract collagen deposition
and portal bridging in Hfe-/-xTfr2mut mice. In addition, there was decreased SOD activity
and enhanced lipid peroxidation in the liver, indicative of increased hepatic oxidative stress
in the Hfe-/-xTfr2mut mouse and to a lesser extent in the Tfr2mut mouse. The evidence of
iron-mediated liver injury seen in the Hfe-/-xTfr2mut mouse is similar to what is reported in
human HH, with mild inflammation, increased collagen deposition and decreased SOD
activity common findings in liver biopsies.
Inflammation has been shown to have a significant effect on iron metabolism causing a
phenomenon known as anaemia of inflammation. Administration of the inflammatory
xii
stimuli, LPS, to HH mice induced inflammation resulting in decreased plasma iron and
NTBI levels with a concurrent increase in liver mRNA expression of the iron importer Zip14
and a decrease in the iron exporter Fpn. Inflammation also increased Hamp1 expression,
though this effect was diminished in HH mice. The increase in Zip14 levels and decrease
in Fpn expression, resulted in liver iron retention consistent with the anaemia of
inflammation, reducing the bioavailability of iron for erythropoiesis. Iron sequestration in
the liver may also contribute to iron-induced liver injury evident in the Hfe-/-xTfr2mut mouse.
In summary, the disruption of HFE and TFR2 resulted in decreased synthesis of the
hepatic iron regulator hepcidin, resulting in elevated plasma iron levels. Excess iron
saturated circulating transferrin, resulting in the presence of NTBI which was rapidly
removed from the circulation and deposited in the liver, kidney, pancreas and heart.
Excess iron in the liver resulted in iron-induced liver injury and fibrosis in mice with
disruption in both HFE and TFR2. Systemic inflammation may also exacerbate iron
sequestration in the liver, enhancing liver iron overload and iron-induced injury in HH.
xiii
Publications and presentations arising from this thesis
Publications
1. Delima RD, Chua AC, Tirnitz-Parker JE, Gan EK, Croft KD, Graham RM, Olynyk
JK, Trinder D. 2012. Disruption of hemochromatosis protein and transferrin
receptor 2 causes iron-induced liver injury in mice. Hepatology 56(2):585-93
Impact factor 11.66.
2. Delima RD, Chua AC, Ho D, Olynyk JK, Trinder D. 2013. In vivo non-transferrin-
bound iron uptake is upregulated in murine models of Hereditary
Haemochromatosis. AJP: Gastrointestinal and Liver Physiology (Under review)
Oral Presentations
1. Delima RD, Chua ACG, Herbison C, Graham R, Olynyk J, Trinder D. 2009.
Disruption of both Hfe and Tfr2 causes more severe hepatic iron overload in
hereditary haemochromatosis; presented at the Hepatology and Luminal Workshop
(Australian Liver Association & Australian Liver Foundation), Yarra Valley,
Australia.
2. Delima RD, Chua ACG, Graham R, Olynyk J, Trinder D. 2010. Disruption of both
Hfe and Tfr2 causes more severe hepatic iron overload in hereditary
haemochromatosis; presented at the American Association for the Study of Liver
Disease annual meeting, Boston, USA.
3. Delima RD, Chua ACG, Ho D, Graham RM, Olynyk JK and Trinder D. 2011. Non-
transferrin bound iron transport in vivo is iron regulated in hereditary
haemochromatosis, presented at presented at the 4th Congress of the International
BioIron Society, Vancouver, Canada.
xiv
List of Figures Page
Figure 1.1: Iron in the human body .................................................................................... 4 Figure 1.2: Model of the pathways of iron transport in the duodenum. ............................... 7 Figure 1.3: Model of iron transport pathways in the hepatocyte. ...................................... 17 Figure 1.4: Model of hepcidin regulatory pathways in the hepatocyte. ............................. 23 Figure 3.1: Plasma iron parameters. ................................................................................ 66 Figure 3.2: Liver metal content ......................................................................................... 70 Figure 3.3: Liver expression of iron regulatory genes. ...................................................... 72 Figure 3.4: Hepatic p-Smad1/5/8 protein expression. ...................................................... 74 Figure 3.5: Liver expression of iron transporter genes. .................................................... 76 Figure 3.6: Duodenal expression of iron transport genes. ................................................ 78 Figure 4.1: Plasma iron parameters. ................................................................................ 92 Figure 4.2: Plasma NTBI clearance ................................................................................. 93 Figure 4.3: Tissue NTBI uptake. ...................................................................................... 94 Figure 4.4: Liver NTBI uptake. ......................................................................................... 96 Figure 4.5: Kidney NTBI uptake. ...................................................................................... 98 Figure 4.6: Pancreas NTBI uptake. .................................................................................. 99 Figure 4.7: Heart NTBI uptake. ...................................................................................... 100 Figure 4.8: Duodenum and femur NTBI uptake .............................................................. 101 Figure 5.1: Hepatic iron concentration. .......................................................................... 115 Figure 5.2: Liver histology. ............................................................................................. 117 Figure 5.3: CD45+ / ferritin double staining in Hfe-/-xTfr2mut mice. ................................... 118 Figure 5.4: Biochemical markers of liver injury. .............................................................. 120 Figure 5.5: Liver collagen deposition via Sirius red stain. ............................................... 122 Figure 5.6: Sirius red stain correlated with iron, collagen and lipid peroxidation
measurement. ........................................................................................... 123 Figure 5.7: Liver collagen deposition using Masson’s trichrome stain. ........................... 124 Figure 5.8: Liver expression of injury-related genes. ...................................................... 126 Figure 6.1: LPS time course. ......................................................................................... 135 Figure 6.2: Liver expression of inflammatory cytokine genes: LPS vs. saline treated mice.
.................................................................................................................. 137 Figure 6.3: Plasma iron parameters: LPS vs. saline treated mice. ................................. 139 Figure 6.4: Liver expression of iron regulatory genes: LPS vs. saline treated mice. ....... 141 Figure 6.5: Liver expression of iron transport genes in LPS and saline treated mice. ..... 143
List of Tables Page
Table 2.1: Reagents used for reverse transcription of RNA. ............................................ 52 Table 2.2: Reagents in the PCR Master Mix. ................................................................... 53 Table 2.3: Primer sequences and annealing temperatures. ............................................. 53 Table 2.4: PCR cycling parameters.................................................................................. 55 Table 3.1: Body and organ weights of HH and WT mice. ................................................. 62 Table 3.2: Haematological parameters in HH and wild type mice. .................................... 64 Table 3.3: Serum markers of liver function in HH and wild type mice. .............................. 68 Table 4.1: Tissue iron content. ......................................................................................... 91
xv
Abbreviations
The following abbreviations are used throughout this thesis:
2-DHBA Dihydroxybenzoic acid 4-HNE 4-Hydroxynonenal ABC ATP-binding cassette ALA Aminolevulinic acid ALAS2 Delta-aminolevulinate synthase 2 ALT Alanine transaminase BCA Bicinchoninic acid BMP6 Bone morphogenic protein-6 BMPR BMP receptor BPS bathophenanthroline disulfonic acid BSA Bovine serum albumin CD45 Cluster of differentiation45 CEBP/α CCAAT/enhancer-binding protein α CHOP CCAAT/Enhancer-Binding Protein Homologous Protein CREB cAMP response element-binding DAPI 4',6-diamidino-2-phenylindole
DcytB Duodenal cytochrome B
DMT1 Divalent metal transporter1 EKLF Erythroid Krüppel-like Factor EPO Erythropoietin ER Endoplasmic reticulum FBXL5 F-box and leucine-rich repeat protein 5 FFPE Formalin fixed paraffin embedded FLVCR Feline leukaemia virus subgroup C cellular receptor FPN Ferroportin GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDF15 Growth differentiation factor 15 gp130 glycoprotein 130 H&E Hematoxylin and eosin HAMP Hepcidin gene Hct Haematocrit HFE Haemochromatosis protein Hfe-/- Hfe knockout mouse HH Hereditary haemochromatosis HIC Hepatic iron concentration HIF-2α Hypoxia inducible factor2α HJV Haemojuvelin HO-1 Haem oxygenase 1 ICP-AES Inductively coupled plasma – atomic emission spectroscopy Id1 Inhibitor of DNA binding-1 IL-6 Interleukin-6 IRE Iron regulatory element IRP Iron regulatory protein ISC Iron-sulfur cluster JAK-STAT Janus kinase-Signal Transducer and Activator of Transcription LAMP1 Lysosomal-associated membrane protein 1 LPS Lipopolysaccharide MCH Mean cell haemoglobin MCHC Mean corpuscular haemoglobin concentration MCV Mean corpuscular volume
xvi
MDA Malondialdehyde MHC Major histocompatability complex MOPS 3-(N-morpholino) propanesulfonic acid MVB Multivesicular bodies NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NMR Nuclear magnetic resonance NO Nitric oxide NTBI Non-transferrin bound iron PBS Phosphate buffered saline PCFT Proton-coupled folate transporter RBC Red blood cell RDW Red blood cell distribution width ROS Reactive oxygen species Scara5 Scavenger receptor class A, member 5 SDS Sodium dodecyl sulphate SLC Solute carrier SOD Superoxide dismutase STEAP Six transmembrane epithelial antigen of the prostate TBA Thiobarbituric acid TBARS Thiobarbituric acid reactive substances TBI Transferrin bound iron TBST Tris buffered saline and Tween TCA Trichloroacetic acid Tfr2mut Tfr2 mutant mouse TFRs Transferrin receptors TGA Thioglycolic acid TGFβ Transforming growth factor β TIBC Total iron binding content TIM2 T cell immunoglobulin and mucin domain 2 TLR4 Toll-like receptor 4 TNF Tumour necrosis factor TWSG1 Twisted gastrulation protein homolog 1 UTR Untranslated region WT Wild-type ZIP14 Zrt- and Irt-like protein 14
2
Chapter 1
Literature review
3
Introduction The unique ability of iron to serve both as an electron donor and acceptor makes it
essential in many physiological and metabolic processes. Fundamental biochemical
activities, such as oxygen transport, energy production and cellular proliferation are all
dependent on iron-containing proteins. Although a necessity at normal levels, excess iron
may become toxic due to its ability to catalyse the production of reactive oxygen species
and damage cellular macromolecules. This is of particular importance in the iron-overload
disorders, Herediatry haemochromatosis (HH) and β-Thalassaemia (Chua et al. 2007).
1.1 Iron in the human body The adult human body contains approximately 3-5 g of iron, with more than two-thirds of
body iron incorporated in the haemoglobin of mature red blood cells and erythroid
precursors (>2 g) (Andrews 1999). The remaining body iron is predominantly found in
macrophages of the reticuloendothelial system (≈600 mg) or within the iron storage
protein, ferritin, in hepatocytes (1000 mg). Small amounts of iron are also present in
myoglobin, in the muscles (≈300 mg) and as a constituent of other cellular iron containing
proteins and enzymes (≈8 mg). In humans, dietary iron absorption is in the range of 1 to 2
mg daily, which is approximately the amount of iron lost by the body each day, through
excretion and blood loss (Fig 1.1), as humans lack a dedicated mechanism for the
excretion of iron (Andrews 1999).
4
Figure 1.1: Iron in the human body Approximately ≈1–2 mg of iron is absorbed daily to account for obligatory losses of a similar amount of iron through sloughing of mucosal and skin cells, haemorrhage, and other losses. Approximately 4 mg of iron is found in circulation bound to Tf, with the majority of body iron found in the erythroid compartment of bone marrow and in mature erythrocytes. Splenic reticuloendothelial macrophages, which recycle iron from senescent red blood cells, provide iron for the synthesis of new red blood cells. Liver hepatocytes store iron in ferritin shells. During pregnancy, 250 mg of iron is transported across the placenta to the foetus.
1.2 Iron absorption The majority of dietary iron is absorbed by the duodenum. There are two forms of dietary
iron: non-haem iron, derived from vegetables and grains, and haem iron, derived from red
meat. The contribution of these two sources of iron varies according to diet. Uptake of iron
occurs predominantly in the enterocytes of the duodenum, with gastric acidity promoting
the chelation of non-haem iron to soluble compounds such as amines, amino acids and
sugars (Hershko et al. 2007). Ninety percent of dietary iron exists as inorganic (non-haem)
5
iron, predominantly in the form of the insoluble oxidised ferric (Fe3+) form. Following
digestion, ferric iron must first be reduced to the ferrous (Fe2+) form by the brush border
ferrireductase, duodenal cytochrome B (Dcytb; (McKie et al. 2001).
1.2.1 Dcytb
The mammalian ferric reductase Dcytb (Fig. 1.2), was first identified in 2001 and was
isolated from hypotransferrinaemic mice (McKie et al. 2001). Dcytb is highly hydrophobic
and has six transmembrane domains and is identical to haem protein p30. It is highly
expressed in the duodenum (McKie et al. 2001) and in erythrocyte membranes (Su et al.
2006), but has also been identified in the liver (McKie et al. 2001), and in airway epithelial
cells (Turi et al. 2006). The expression of Dcytb is strongly regulated by iron with Dcytb
mRNA and protein levels up-regulated in iron-deficient duodenum (Frazer 2002). However,
iron status does not appear to modulate Dcytb expression in the liver and spleen (McKie et
al. 2001). Regulation of Dcytb is hypothesised to be controlled by yet unidentified
transcription factors similar to the iron-inducible transcriptional regulator, Aft1p, which
controls yeast ferric reductases, but has been shown to be a direct regulatory target of
Hypoxia Inducible Factor 2α (HIF-2α) in response to hypoxic conditions (Mastrogiannaki et
al. 2009; Shah et al. 2009). Though the disruption of Dcytb in mice did not significantly
alter body iron stores (Gunshin et al. 2005), studies injecting radioactive 59Fe into tied
duodenal segments, demonstrated decreased iron uptake into the mucosa of Dcytb-
knockout mice (Choi 2008), supporting the role of Dcytb in the reduction of dietary iron.
Once reduced, the ferrous iron is then transported across the cell membrane by divalent
metal transporter 1 (DMT1; Figure 1.2) (Gunshin et al. 1997).
1.2.2 DMT1
The human DMT1 gene consists of 167 exons spread over more than 36 kb (Lee 1998).
The DMT1 protein is highly hydrophobic, with twelve predicted transmembrane domains
and both the amino-terminus and carboxy-terminus are predicted to exist within the
6
cytoplasm (Zhou et al. 1998). DMT1 is capable of transporting a variety of divalent metals
including copper (Arredondo et al. 2003), cobalt, cadmium (Picard et al. 2000), and lead
(Garrick et al. 2006). It has also been reported that DMT1 can co-transport protons
(Gunshin et al. 1997) and functions optimally at approximately pH 6 (Su 1998). The
expression of intestinal DMT1 has been shown to be inversely regulated by iron status via
a post-transcriptional mechanism (See section 1.8.3).
1.2.3 Haem uptake
In most non-vegetarian diets more than one-third of the total daily iron is supplied by
dietary haemoglobin and myoglobin (Carpenter 1992). Before haem iron can be utilised,
haem must be cleaved from the haemoglobin and myoglobin proteins by proteolytic activity
in the lumen of the stomach and small intestine (Conrad 1967). Haem binds to the brush
border membrane of duodenal enterocytes and is translocated across the membrane by a
yet to be identified haem transporter (Figure 1.2). The transporter Haem Carrier Protein 1
(HCP1) was initially identified as a putative haem transporter (Shayeghi et al. 2005),
however its low affinity for haem (Km 125µM) and subsequent studies demonstrating its
high affinity for folate and its derivatives, has resulted in HCP1 being re-identified as
Proton-Coupled Folate Transporter (PCFT) (Qiu et al. 2006). After transport across the
apical membrane, the haem is degraded by haem oxygenase to release the ferrous ion
(Raffin 1974), which enters the low-molecular-weight iron pool in the enterocyte. The iron
may be stored in the cell as ferritin or transported across the basolateral membrane to the
plasma. Iron export across the basal membrane of the enterocyte is dependent on the iron
exporter, ferroportin, and the ferroxidase, hephaestin, which converts Fe2+ to Fe3+ and is
then bound by circulating transferrin (Fig. 1.2) (Donovan et al. 2005).
7
Figure 1.2: Model of the pathways of iron transport in the duodenum. Uptake of ionic iron and haem iron from the lumen into the enterocyte across the apical membrane and transport out across the basolateral membrane to the blood (Chua et al. 2007). DMT1: Divalent metal transporter 1. DCYTB: Duodenal cytochrome b. FPN: Ferroportin. TF: Transferrin.
1.3 Iron export
1.3.1 Ferroportin
To date, only one iron exporter has been identified: ferroportin (FPN) (Donovan et al.
2005). FPN also known as Ireg1 (McKie et al. 2000) and Mtp1 (Abboud and Haile 2000)
was initially identified as an iron-export protein located on the basolateral membrane of
enterocytes (Figure 1.1). Subsequently it has been shown that FPN is a ubiquitously
expressed cell surface protein with 12-predicted transmembrane domains (Canonne-
Hergaux et al. 2006). Studies using Xenopus oocytes have shown that over-expression of
FPN results in an increase in iron released from the cells accompanied by a reduction in
ferritin expression, indicating a reduction of intracellular iron levels (Chung 2003). FPN
mRNA has been found to be highly expressed in duodenal enterocytes, spleen, kidney,
and liver, particularly in Kupffer cells and to a lesser degree in hepatocytes (Philpott 2002).
FPN expression is regulated at a transcriptional level by hypoxia (McKie et al. 2000),
Fe2+
Fe3+
Ferritin
Fe2+
Fe
2+
Fe2+
Fe2+
Gut Lumen Enterocyte Blood
DMT1
FPN
Hephaestin
Haem Haem
Haem
Oxygenase
DCYTB
Haem
transporter
Transit Iron Pool
Non-haem
iron Fe3+
Tf
Fe2Tf
Fe2+
DMT1
Fe2+
8
inflammation (Yang et al. 2002), and haem, and iron (Knutson et al. 2003) and zinc
(Troadec et al. 2010) concentration. FPN is also post-transcriptionally regulated by cellular
iron levels via a Iron Responsive Protein (IRP)/Iron Responsive Element (IRE) post-
transcriptional mechanism (Abboud and Haile 2000)(see Section 1.8.3). Finally, FPN is
also regulated via a post-translational hepcidin-dependent mechanism (Nemeth et al.
2004)(see Section 1.8.4) and a hepcidin-independent substrate-dependent mechanism
(De Domenico et al. 2011). Once the iron is released from the cell, it is oxidised by
multicopper ferroxidases: hephaestin at the basolateral surface of the enterocyte or by
caeruloplasmin in other types of cells (De Domenico et al. 2007).
The expression of FPN at the cell surface is regulated by the antimicrobial peptide,
hepcidin, which through the interaction of hepcidin and duodenal FPN inhibits iron release
from the enterocyte and subsequently reduces plasma iron levels (see Section 1.8.4;
(Nemeth et al. 2004). First isolated from blood (Krause et al. 2000) and then urine (Park et
al. 2001), hepcidin expression was found to be regulated by body iron levels, with mice
deficient in hepcidin developing iron overload (Nicolas et al. 2001) and mice over-
expressing hepcidin developing severe anaemia and iron deficiency (Nicolas et al. 2002)
Though the regulation of FPN by hepcidin is well documented in macrophages, recent
studies suggest that this is a cell-specific effect, as numerous studies suggest that in the
duodenum, hepcidin may interact with the iron transporter DMT1 (Yamaji et al. 2004;
Chaston et al. 2008; Brasse-Lagnel et al. 2011).
Iron not released from the enterocyte is eventually lost through cell sloughing. FPN is also
highly expressed in macrophages and plays a key role in iron recycling. Senescent red
blood cells are phagocytosed by the macrophages of the reticuloendothelial system
(Kondo et al. 1988) Macrophages degrade haemoglobin and catabolise haem via haem
oxygenases (HO-1 and HO-2), liberating inorganic Fe2+, before being exported from the
9
cell by FPN and re-oxidised by caeruloplasmin, where it binds to the iron transport protein,
transferrin (Fig. 1.2), for delivery to various tissues.
1.4 Plasma iron
1.4.1 Transferrin-bound iron
Transferrin is a monomeric, iron-binding glycoprotein composed of two structurally similar
lobes, each containing a single iron-binding site (Lambert 2012). It is expressed
predominantly in the foetal and adult liver and is transcriptionally regulated by iron status
with iron deficiency resulting in a 2-4 fold increase in the rate of transferrin synthesis
(Theisen et al. 1993). Low amounts of transferrin are also synthesised by other tissues,
such as the brain and testis (Zakin 1992). Plasma transferrin is a powerful chelator,
capable of binding iron tightly but reversibly (Huebers and Finch 1987). A molecule of
transferrin can potentially bind two atoms of Fe3+ with high affinity, which is higher in the
extracellular pH of 7.4 and decreases in the acidified endosomes, allowing for the
dissociation of the Fe3+. Iron bound to plasma transferrin accounts for less that 0.1% (≈3
mg) of total body iron but represents the most active iron pool in the body. More than 2
million erythrocytes are produced every second by the bone marrow, requiring a daily
supply of at least 20-30 mg of iron. To meet the requirements of erythropoiesis, plasma
transferrin turns over more than 10 times a day. It has been calculated that atoms of iron
entering the plasma transferrin pool will remain in the circulation for only 90 minutes before
being taken up by the bone marrow (Cavill 2002), with more than 80% being incorporated
into erythroblasts (Ponka et al. 1998). The saturation of transferrin with iron is an indicator
of body iron stores, but also reflects the balance between dietary iron absorption,
reticuloendothelial iron release and uptake by the bone marrow. Under normal conditions,
approximately 30% of the transferrin iron-binding sites are saturated. Low transferrin
saturation in conjunction with the high-affinity for iron, allows transferrin to efficiently buffer
10
alterations in plasma iron levels, removing free iron from the circulation and minimising its
potential toxicity. In humans, a transferrin saturation of less than 15% indicates iron
deficiency, whereas saturations greater than 45% may indicate iron overload (Hentze et al.
2010). In disorders of iron overload, where transferrin saturation exceeds 60%, levels of
the toxic, redox-active non-transferrin bound iron (NTBI) increases dramatically (to 5 µM
and even higher) leading to possible tissue damage (Craven et al. 1987).
1.4.2 Non-transferrin bound iron
NTBI is a low-molecular-weight form of iron that is thought to play a major role in
pathological iron overload, via its ability to catalyse the formation of reactive oxygen
species (Jomova and Valko 2011). Despite its importance in the pathophysiology of iron
overload, the exact chemical composition of NTBI is still poorly understood. In the blood
plasma citrate, acetate, pyruvate and phosphates are all potential ligands, but is
considered to be the most likely ligand (May 1977), with 1H NMR studies on the serum of
haemochromatosis patients conclusively demonstrating the involvement on citrate in NTBI
coordination (Grootveld et al. 1989).
1.4.3 Transferrin-bound iron uptake
1.4.3.1 Transferrin Receptor 1 (TFR1)
Nearly all cells acquire iron via the uptake of transferrin-bound iron mediated by transferrin
receptors (TFRs). There are two types of TFRs, known as TFR1 and TFR2 present on cell
membranes. TFR1 is expressed by most types of cells except mature erythrocytes and
TFR2 is predominantly expressed by hepatocytes and erythroid precursors. TFR1 consists
of two identical glycoprotein transmembrane subunits linked by a disulphide bond. The
efficiency of TFR1 to deliver transferrin-bound iron depends on the iron content of the
transferrin, with differic transferrin binding to TFR1 with an affinity 30- and 500-fold higher
than mono-ferric and apo-transferrin, respectively (Young et al. 1984). Each receptor
subunit is capable of binding one molecule of transferrin, thus TFR1 can bind 2 molecules
11
of transferrin. The dimeric TFR1 can mediate the uptake of four atoms of iron if each
molecule is saturated with iron (Sheth 2000). The expression of TFR1 is regulated by
cellular iron levels by a post-transcriptional mechanism (Section 1.8.3). TFR1 expression
is inversely regulated by cellular iron levels; low iron upregulates TFR1 and high iron
downregulates TFR1 levels. This regulatory process ensures the iron supply meets the
iron requirements of the cell and occurs at the post-transcriptional level through the
binding of iron regulatory proteins (IRP) to the iron regulatory elements (IRE) present in
the 3’ untranslated region of the TFR1 mRNA (Section 1.8.3) (Muckenthaler et al. 2008).
TFR1 is upregulated by cytokines, such as interleukin-2 (Seiser et al. 1993), mitogens
(Ouyang et al. 1993) and growth factors (Miskimins et al. 1986), as well as via hypoxia-
inducible factor 1α (HIF-1α), which binds to a conserved hypoxia regulatory element
binding site within the TfR1 promoter (Tacchini et al. 1999).
TFR1 mediated uptake of diferric transferrin has been studied extensively (Graham et al.
2007). Initially it involves the high-affinity interaction of differic transferrin with TFR1 at the
cell surface at pH 7.4 (Morgan 1981). Formation of the transferrin and receptor complex
triggers internalisation of the complex into a clathrin-coated vesicle (Harding 1983) which
matures into a proton-pumping endosome (Fig. 1.3). As the pH of the endosome
approaches 5.5, a conformational change in transferrin results in the release of the iron
(Klausner et al. 1983) and the increased binding affinity of transferrin for TFR1. The iron
released from transferrin into the endosome is reduced by the ferrireductase, six-
transmembrane epithelial antigen of the prostate (STEAP) 3 before being exported to the
cytoplasm via either DMT1 (Ohgami et al. 2005) or the zinc transport protein, Zrt- and Irt-
like protein-14 (ZIP14) (Fig. 1.3) (Zhao et al. 2010). The iron enters the liable iron pool and
is used immediately for cellular processes or stored in the iron storage protein, ferritin. The
acidified compartment containing the iron-depleted transferrin (apo-transferrin) still bound
to TFR1 returns to the cell surface where at a pH of 7.4 due to a decrease in binding
12
affinity, the apo-transferrin is released from the receptor to the circulation. This allows the
TFR1 to bind more diferric transferrin and the transferrin-cell cycle to continue and deliver
more iron to the cell. It has been shown that iron delivery via the transferrin-TFR1 cycles
can be completed in approximately 5-20 min depending on cell type (Morgan and Baker
1986; Qian and Morgan 1992).
Although the high-affinity transferrin-TFR1 pathway is the predominant mechanism by
which most cells take up transferrin-bound iron, studies in Huh7 hepatoma cells suggest
the existence of internalisation of transferrin-bound iron via a low-affinity, TFR1-
independent manner (Trinder et al. 1996). At low transferrin concentrations, less than 0.3
µmol/L, transferrin-bound iron uptake occurs predominantly through TFR1, whilst at higher
transferrin concentrations, low-affinity mechanisms predominate (Anderson et al. 1994).
Interestingly, the physiological concentration of differic transferrin in human plasma is ≈5
µmol/L (Ponka et al. 1998), suggesting that in many cell types the low-affinity TFR1-
independent pathway dominates the acquisition of transferrin-bound iron. The exact
mechanisms of TFR1-independent uptake of transferrin-bound iron are still not clearly
characterised, however, one possible mechanism may involve transferrin receptor 2
(TFR2).
1.4.3.2 Transferrin Receptor 2 (TFR2)
TFR2 is a 105 kDa membrane protein with significant homology to TFR1. The TFR2 gene
expresses two transcripts, an alpha form (2.9 kb) and a beta form (2.5 kb). The TFR2-α
protein is a type-II membrane protein that has 45% identity and 66% similarity with the
extracellular domain of TFR1, and is most significantly expressed by the liver, with much
lower expression in the spleen, lung, muscle, prostate (Kawabata et al. 1999) and in early
erythroid precursors (Kawabata et al. 2001). The TFR2-β protein has been shown to lack
the amino-terminal portion of the TFR2-α protein including the putative transmembrane
13
domain. However its expression is much more common than TFR2-α with significant
expression not only in the liver, but also in the brain, heart and spleen (Kawabata et al.
1999).
Though significantly homologous to TFR1, TFR2 has a 30-fold lower affinity for differic
transferrin than TFR1 (West et al. 2000). In addition, TFR2 protein levels show a dose-
dependent response to transferrin saturation, with increasing concentrations of diferric-
transferrin increase receptor levels by stabilising TFR2 protein (Johnson and Enns 2004;
Robb and Wessling-Resnick 2004; Chen and Enns 2007). TFR2 is not regulated by
intracellular iron levels (Kawabata et al. 2001) unlike TFR1, which is inversely regulated by
cellular iron status via the posttranscriptional iron responsive element-iron regulatory
protein (IRE-IRP) mechanism (Section 1.8.3). TFR2 is also unable to compensate for the
loss of TFR1 function, as Tfr1-knockout mice display embryonic lethality, and TfR1+/− mice
have lower hepatic iron levels than wild-type mice (Kawabata et al. 2000), this in contrast
to the hepatic iron overload associated with human or murine mutations in TFR2
(Camaschella et al. 2000).
The uptake of iron-loaded transferrin by TFR2 in liver Huh-7 cells and rat hepatocytes is
characterised by a pattern of biphasic internalisation kinetics. With steady-state
internalisation of transferrin typically saturated at low concentrations (<0.5 µM) (Blight and
Morgan 1983), this atypical pathway displays a linear phase of uptake at ligand
concentrations >0.5 µM, the subcellular distribution of the internalised ligand indicated that
TFR2 delivers transferrin to the late endocytic pathway (Figure 1.2), where it accumulated
in multivesicular bodies (MVB) devoid of receptors and deficient in lysosome-associated
membrane protein 1 (LAMP1), resulting in transferrin not being degraded (Robb and
Wessling-Resnick 2004). This suggests a model in which the apparently nonsaturable
14
linear phase of transferrin uptake by TFR2 can be explained by the intracellular
accumulation of transferrin.
However, as TFR2 tissue distribution is limited, it is unlikely that TFR2 plays a major role in
low-affinity, TFR1-independent iron uptake, with only a minor decrease in hepatic
transferrin-bound iron uptake reported in TFR2 mutant mice (Chua et al. 2010). Other
potential mediators for low-affinity TFR1-independent uptake of transferrin-bound iron
include cubulin, which operates conjugated to its co-receptor, megalin (Kozyraki et al.
2001), in the proximal tubules of the kidney. Cubulin is expressed on the apical membrane
of proximal tubule cells and mediates the re-absorption of filtered transferrin from the
glomerular filtrate (Smith and Thevenod 2009). Other mechanisms include endocytosis
mediated by glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in macrophages (Raje
et al. 2007) and proteoglycans in hepatocytes (Hu and Regoeczi 1992).
1.4.4 Non-transferrin bound iron uptake
A small fraction (<1%) of iron in the plasma circulates as a non-transferrin form conjugated
to small molecules such as citrate and amino acids. In cases of physiological iron overload
circulating transferrin binding sites are saturated and excess iron or NTBI may be found in
the plasma. NTBI is extremely toxic due to its potential to generate free radicals that can
damage essential biological molecules and it is rapidly cleared from the plasma by the liver
(Brissot et al. 1985). Studies have shown that, at least in the rat, more than 60% of NTBI is
removed from the circulation on its first past through the liver at a rate of approximately 30
nmol/min/g liver, whilst in the same experiment less than 1% of transferrin-bound iron was
cleared by the liver on its first pass (Wright et al. 1986). A subsequent study suggests that
the plasma half-life of NTBI may be less than 30s (Craven et al. 1987). These studies
indicate that the liver is part of an extremely efficient mechanism for the removal of NTBI
from the circulation, however the exact mechanism by which this occurs is still poorly
understood. DMT1 was initially proposed to be the major NTBI transporter in the liver
15
(Figure 1.2), the principal site of NTBI deposition. However, liver-specific Dmt1-/- mice are
not protected against hepatic iron accumulation (Gunshin et al. 2005). This is consistent
with the knowledge that limited iron transport occurs by proton-dependent DMT1 at the
neutral pH of the extracellular fluid. These findings clearly suggest the existence of other
NTBI uptake pathways.
1.4.4.1 Zrt- and Irt-like Protein 14 (ZIP14)
Although its role in liver iron-loading remains to be established, ZIP14 has been shown to
facilitate the uptake of NTBI in cultured cells (Liuzzi et al. 2006). ZIP14 is a member of a
large family of metal-ion transporters, the SLC39 family (Lichten and Cousins 2009). ZIP14
is abundantly expressed in the liver, heart and pancreas (Liuzzi et al. 2006), the major
sites of tissue injury in iron overload. Further evidence for the involvement of ZIP14 in
NTBI transport (Figure 1.2) comes from studies that have utilised siRNA to reduce ZIP14
protein levels and in turn reduce iron and zinc uptake (Liuzzi et al. 2006). In the rat liver
perfusion model, it has been shown that zinc strongly inhibited the uptake of ferrous iron
(Wright et al. 1986). Similar observations have been made in primary hepatocytes from
mice (Chua et al. 2004) and rats (Baker et al. 1998), where the presence of zinc inhibited
the uptake of ferric citrate. ZIP14 has been shown to be specific for Fe2+ with Fe3+ not
transported (Pinilla-Tenas et al. 2011). Though most NTBI in the plasma exists as Fe3+-
citrate, a significant amount may be reduced to Fe2+ via the presence of plasma L-ascorbic
acid (May et al. 1999), superoxide (Ghio et al. 2003), the presence of mammalian cell
surface ferrireductases in hepatocytes and other cell types (Inman et al. 1994; Jordan and
Kaplan 1994), or by citrate chelating Fe2+ in addition to Fe3+.
NTBI can also be taken up by cardiomyocytes via L-type voltage-dependent calcium
channels. When mice with iron overload were treated with calcium channel blockers, there
was an attenuation of myocardial iron accumulation and a decrease in iron-associated
16
oxidative stress (Oudit et al. 2003). A similar role for voltage-gated calcium channels has
been proposed for the delivery of iron to neuronal cells (Gaasch et al. 2007).
In the kidney, iron-loaded lipocalin 2 can be endocytosed by the receptors 24p3R
(Devireddy et al. 2005) and megalin (Hvidberg et al. 2005). These mechanisms for the
uptake of NTBI primarily operate during organ development (Yang et al. 2002), injury (Mori
et al. 2005), or inflammation (Devireddy et al. 2005). However, as lipocalin 2 deficient mice
do not exhibit any observable defects of iron metabolism (Flo et al. 2004), this excludes
lipocalin 2 from functioning as a major mechanism for iron uptake.
1.4.4.2 Ferritin uptake
Serum ferritin has also been implicated in the uptake of NTBI, particularly in pathological
conditions in which tissue damage results in the release of iron dense intracellular ferritin
into the plasma. The endocytosis of ferritin is mediated by a ferritin receptor and several
candidate molecules have been identified including T-cell immunoglobulin-domain and
mucin-domain 2 (TIM-2) in cells of the spleen, liver, bile duct, and kidney (Chen et al.
2005) and Scavenger receptor class A, member 5 (Scara5) in epithelial cells (Li et al.
2009). Studies have also shown that the H-subunit of ferritin can be internalised upon
specific binding to TFR1 (Li et al. 2009).
1.4.4.3 Haem/Haemoglobin uptake
A further mechanism of NTBI uptake occurs when intravascular haemolysis results in the
release of haemoglobin or free haem into the plasma. The liver-derived plasma protein
haptoglobin binds free haemoglobin and promotes its endocytosis by macrophages, upon
recognition and binding to the CD163 receptor (Kristiansen et al. 2001). Similarly,
haemopexin scavenges free haem and the resulting complex is endocytosed via the CD91
receptor (Figure 1.3) present on the surface or macrophages, hepatocytes and possibly
other cell types (Hvidberg et al. 2005).
17
Figure 1.3: Model of iron transport pathways in the hepatocyte. Uptake of transferrin bound iron (Transferrin receptors 1 and 2: TfR1 and TfR2), non-tranferrin bound iron (Divalent metal transport 1: DMT1 and Zrt- and Irt-like protein 14: ZIP14), and haem (Cluster of differentiation 91 receptor: CD91R). Export of iron from the endosome (DMT1, Six transmembrane epithelial antigen of the prostate 3: STEAP3, and ZIP14) and iron release from the cell (Ferroportin and caeruloplasmin)(Figure modified from (Graham et al. 2007).
1.5 Cellular iron Once iron leaves the endosome, it becomes part of a transient cytosolic pool of iron,
presumably bound to low molecular weight intracellular chelators, such as citrate, various
peptides, ATP, AMP or pyrophosphate, which is known as the labile iron pool (Kakhlon
and Cabantchik 2002). Although it represents only a small fraction total cellular iron (≈3-
5%), the cytosolic labile iron pool is a reflection of cellular iron status (Kruszewski 2003),
with fluctuations triggering homeostatic adaptive responses.
18
Cytosolic iron can enter the mitochondria via the SLC transporter mitoferrin, which is
localised to the inner mitochondrial membrane (Shaw et al. 2006). Studies also suggest
that 2,5-dihydroxybenzoic acid (2-DHBA), a ligand for lipocalin 2, may also play a role in
mitochondrial iron transport (Devireddy et al. 2005), as cells unable to synthesise this
mammalian siderophore accumulate excessive quantities of cytoplasmic iron. However,
there is still some controvery regarding this work has it has been difficult to reproduce
(Correnti et al. 2012).Further studies utilising erythroid cells suggest that iron may also be
transported directly from the endosomes to the mitochondria via a “kiss and run”
mechanism involving direct contact between the two organelles (Richardson et al. 2010).
Once within the mitochondria, iron is primarily used for the synthesis of haem and iron
sulphur clusters (ISCs).
1.5.1 Haem synthesis
The synthesis of haem from the precursor 5-aminolevulinic acid (ALA) occurs via an
evolutionarily conserved eight step pathway. Briefly, ALA is exported to the cytosol where
it is converted into the intermediate metabolites porphobilinogen, hydroxymethylbilane,
uroporphyrinogen III and copro-porphyrinogen III, which is oxidised to protoporphyringen
IX and imported into the mitochondria where it is further oxidised to protoporphyrin IX. In
the final step of the haem synthesis pathway, ferrochelatase catalyses the insertion of Fe2+
into protoporphyrin IX. The newly synthesised haem is then exported to the cytosol for
incorporation into haem-containing proteins (Ryter and Tyrrell 2000). It is suggested that
the transport of haem and its intermediates across mitochondrial membranes might involve
the ATP-binding cassette (ABC) family of transporters (Severance and Hamza 2009) and
the SLC transporter, SLC25A39 (Nilsson et al. 2009). In non-erythroid cells, the rate-
limiting step of haem synthesis is the production of ALA. This is in contrast to what occurs
in erythroid cells, where it is the synthesis of the porphyrin ring, a step that is iron-
dependent, that is rate limiting (Ponka 1997). Interestingly, the enzyme ALA synthase,
responsible for the production of ALA in erythroid precursor cells (Srivastava et al. 1988),
19
contains an Iron Response Element and as described in section 1.8.3 is post-
transcriptionally regulated by Iron-Responsive Proteins, providing another point of
regulation for the haem biosynthesis pathway.
1.6 Iron storage
1.6.1 Ferritin
Cells may export excess intracellular iron by exporting Fe2+ via ferroportin or by secretion
of haem through the putative haem exporter, feline leukaemia virus, subgroup C, receptor
(FLVCR) (Keel et al. 2008). However, cells can also store and detoxify excess intracellular
iron in the cytosol by incorporating it into the iron storage protein, ferritin. Ferritin is able to
store large amounts of iron in a soluble, non-toxic form.
Synthesis of ferritin in the liver occurs primarily on free polyribosomes within the
hepatocyte (Konijn 1977) with the rate of ferritin production regulated by the iron status of
the cell by a post-transcriptional mechanism (Section 1.8.3). Ferritin is composed of an
apoprotein shell with a molecular weight of approximately 480 kDa which surrounds a core
of up to 4500 atoms of iron in the form of the mineral ferrihydrite (Harrison 1987). The
ferritin molecule is composed of 24 subunits of two structurally distinct subunit types. The
heavy or H-subunit has a more acidic isoelectric point and a molecular weight of
approximately 21 kDa and the light of L-subunit has a more basic isoelectric point and a
molecular weight of approximately 19 kDa. Differing proportions of each subunit give rise
to isoferritins which have characterisitic isoelectric points and tissue distribution. These
isoferritins (L- and H-type ferritin) are functionally different, with H-ferritin taking up iron
more rapidly than L-ferritin and storing iron in a more metabolically available form
(Harrison and Arosio 1996). In addition, H-type ferritin has biological effects that are
unrelated to iron binding, such as the capacity to inhibit cell growth (Guo et al. 1998),
20
lymphocyte proliferation (Morikawa et al. 1995) and inflammatory signalling (Ramm and
Ruddell 2010). Non-parenchymal liver cells, mainly Kupffer and endothelial cells, of iron-
loaded rats, contained the acidic H-type isoferritin that is not present in hepatocyte ferritin
(Doolittle and Richter 1981).
Iron stored within ferritin is bioavailable and may be mobilised for metabolic purposes
during its lysosomal turnover (Zhang et al. 2010) and possibly also following structural
rearrangements of the ferritin subunits (Takagi et al. 1998). The induction of ferroportin
promotes mobilisation and export of ferritin-derived iron, followed by mono-ubiquitination
and degradation of the apo-ferritin by the proteasome (De Domenico et al. 2006).
1.6.2 Haemosiderin
With normal levels of cytosolic iron, soluble iron-containing ferritin is present in the cytosol
as randomly dispersed ferritin particles. As cytosolic iron levels increase, as in conditions
of pathological iron overload, the concentration of dispersed ferritin increases and small
clusters of ferritin particles appear, still soluble and spread throughout the cytosol. With
further increases in cytosolic iron, ferritin is collected in lysosomes by fusion of ferritin
clusters with lysosomal membranes or by autophagocytosis (Iancu 1992). It is suggested
that digestion of ferritin within secondary lysosomes (siderosomes) leads to denaturation
of ferritin protein subunits (Miyazaki et al. 2002) and to the aggregation of the ferritin iron
cores, resulting in the formation of amorphous, insoluble masses known as haemosiderin
(Iancu et al. 1997). Haemosiderin, which is most commonly found in macrophages, is
especially abundant in situations following haemorrhage but is also found in pathological
conditions in which the capacity of ferritin to store iron is overwhelmed. Ferritin iron may
have a pro-oxidant role and contribute to tissue damage (Arosio and Levi
2010). Production of haemosiderin, enclosed within siderosome membranes, may
sequester the excess iron away from the cytosol and help protect against iron toxicity. As
the total amount of tissue iron increases, the proportion stored as haemosiderin rises, from
21
trace amounts in normal individuals to 90% or more in patients with severe iron overload
(Selden et al. 1980).
Depending upon cell type, iron supply and utilisation, the half-life of cellular ferritin may
range from ≈20 to 96 h (Truty et al. 2001) with haemosiderin having a much slower cellular
turnover than ferritin.
1.7 Systemic Iron homeostasis In mammals iron homeostasis is regulated at the level of iron absorption as there is no
physiological pathway for the excretion of iron. Impaired regulation of iron absorption leads
to either iron deficiency or overload. This complex task is accomplished by the liver-
derived peptide hormone, hepcidin, that responds to multiple regulatory signals, including,
iron availability, erythropoietic activity, inflammation, endoplasmic reticulum (ER) stress
and hypoxia (Hentze et al. 2010).
1.7.1 Hepcidin
Activated hepcidin is a 25 amino acid peptide (Park et al. 2001), synthesised primarily by
hepatocytes, but also at significantly lower levels in other cell types, upon cleavage of the
larger 84 amino acid pro-peptide by the pro-hormone convertase, furin (Valore and Ganz
2008). Mature hepcidin is secreted into the plasma and circulated bound to α2-
macroglobulin (Peslova et al. 2009). Hepcidin levels increase in response to iron (Pigeon
et al. 2001), ER stress (Vecchi et al. 2009), or inflammation (Nemeth et al. 2004). In
contrast, it has been shown that hepcidin levels decrease with iron deficiency, hypoxia and
increased erythropoiesis (Nicolas et al. 2002).
22
1.7.2 Iron-dependent -hepcidin signalling
1.7.2.1 BMP/SMAD signalling
It is proposed that hepatic iron content induces hepcidin expression via bone morphogenic
protein (BMP) signalling (Babitt et al. 2006). Activation of the BMP receptor by BMP
binding (Steinbicker et al. 2011) leads to phosphorylation of the intracellular SMAD1/5/8
(mothers against decapentalegic; (Kautz et al. 2008). Subsequently, p-SMAD1/5/8 forms a
complexe with SMAD4 before translocating to the nucleus, where upon it binds to two
BMP-responsive elements (BMP-RE 1 and 2) at proximal and distal sites of the hepcidin
promoter, thereby inducing its transcription (Fig. 1.4)(Casanovas et al. 2009)) The
aforementioned model is validated by experiments in which SMAD4-/- mice have
decreased hepcidin expression and develop iron overload (Wang et al. 2005) and by
experiments that show that inhibitory SMAD7 prevents the interaction of pSMAD1/5/8 with
SMAD4 resulting in decreased hepcidin expression (Mleczko-Sanecka et al. 2010). BMP2,
5, 6, 7 and 9 have all been shown to induce hepcidin expression in vitro, however, BMP6
is the most physiologically relevant, with liver-specific disruption of BMP6 resulting in
nearly undetectable hepcidin levels with concomitant iron overload, excluding a
compensatory role for the other BMPs (Andriopoulos et al. 2009). The mechanism by
which BMP6 is induced by iron is still not fully understood, but as BMP6 mRNA levels have
been shown to correlate with hepatic iron content, it would suggest a role for BMP6 in
sensing alterations to hepatocellular iron (Kautz et al. 2008).
1.7.2.2 Haemojuvelin
Haemojuvelin (HJV) is a BMP co-receptor that can complex with Type I and II BMP
receptors, enhancing BMP/SMAD signal transduction and increasing hepcidin expression
(Figure 1.4)(Babitt et al. 2006). HJV is predominantly expressed in hepatocytes and
skeletal muscle and is bound to cell membranes via a C-terminal Glycophosphatidylinositol
anchor. Studies suggest that membrane-bound hepatocellular HJV can be inactivated
following cleavage by matriptase-2, thereby decreasing BMP/SMAD signalling and
23
negatively regulating hepcidin expression (Silvestri et al. 2008). In genome-wide
association studies, mutations in matriptase-2 have been shown to correlate with serum
iron, transferrin saturation (Tanaka et al. 2010) and haemoglobin levels (Chambers et al.
2009). Expression of matriptase-2 has also been shown to be up-regulated by hypoxia and
BMP6 or iron, with BMP6 most likely working via induction of Id1 (inhibitor of DNA binding
1) (Meynard et al. 2011). Haemojuvelin cleaved my matriptase-2 results in soluble HJV
which has also been shown to directly compete with BMP6 for binding to the BMPR (Nili et
al. 2010), resulting in decreased BMP/SMAD signalling.
Figure 1.4: Model of hepcidin regulatory pathways in the hepatocyte. The figure shows the hepcidin regulation pathways (Receptor-regulated SMAD: R-SMAD; SMAD1/5/8, Janus kinase 1: JAK1, Signal transducer and activator of transcription 3: STAT3, and phophorylation: P) by which differic transferrin (Transferrin receptors 1 and 2: TfR1 and TfR2, Haemochromatosis protein: HFE), intracellular iron levels (Bone morphogenic protein 6: BMP6, BMP6 receptor: BMP6R, and Haemojuvelin: HJV) and inflammation (Interleukin 6: IL6, IL6 receptor: IL6R, and glycoprotein 130: gp130) regulate hepcidin mRNA.
24
1.7.2.3 HFE/TFR2
The regulation of hepcidin occurs via two pathways, intracellular iron concentration affects
hepcidin signalling via the BMP/SMAD pathway and via a more dynamic pathway, plasma
transferrin saturation affects hepcidin via the HFE/TFR2. Haemochromatosis protein (HFE)
is an MHC-1 like molecule, and as such, interacts with β2-microglobulin (β2M; (Feder et al.
1996). The observation that loss of β2M leads to iron overload was the first suggestion that
linked HFE to iron metabolism (Porto et al. 1998). Subsequently, TFR1 (Parkkila et al.
1997) and its homologue TFR2 (Goswami and Andrews 2006) were also identified as
binding partners of HFE. Even though TFR1 and TFR2 have similar structures, it has been
shown that the domains through which they interact with HFE are different, since the
transferrin binding site of TFR2 does not overlap with the HFE binding site as it does with
TFR1 (West et al. 2001). It is proposed that when differic-transferrin binds to TFR1, HFE
dissociates and binds to TFR2, which has been shown to be capable of binding differic
transferrin and HFE simultaneously (Chen et al. 2007). In addition, it has been
demonstrated that TFR2 will compete with TFR1 for HFE binding regardless of differic
transferrin levels (Goswami and Andrews 2006), and that the HFE/TFR2 complex is
necessary for the induction of hepcidin expression (Fig. 1.4). However, recent studies
utilising combined Hfe and Tfr2 disruption in mice show that these animals develop more
severe iron overload than their single mutant counterparts, suggesting that HFE and TFR2
may exert some independent effects on hepcidin regulation (Wallace et al. 2009; Corradini
et al. 2011). The HFE/TFR2 complex is proposed to signal to hepcidin via the ERK/MAP
(extracellular signal regulated kinase/mitogen activated protein) kinase pathway (Fig. 1.4),
as this pathway has been shown to be activated when cultured murine hepatocytes are
treated with differic transferrin, with subsequent treatment with an ERK inhibitor blocking
the induction of hepcidin by diferric transferrin (Ramey et al. 2009). Similarly, Hfe-/-, Tfr2-/-,
and Hfe-/-xTfr2-/- mice all show significant decrease in hepatic p-ERK1/2 levels, suggesting
that both HFE and TFR2 signal hepcidin via the ERK1/2 pathway (Wallace et al. 2009).
25
1.7.3 Erythropoietic Hepcidin signalling
Studies have shown that the injection of erythropoietin (EPO) in mice (Pak et al. 2006) and
the treatment of isolated mouse hepatocytes with EPO (Pinto et al. 2008) resulted in a
dose-dependent decrease in hepcidin. In addition, human volunteers treated with EPO
have greatly reduced urinary hepcidin levels (Robach et al. 2009). EPO has been shown in
vitro to directly suppress hepcidin expression, decreasing the binding of CEBP/α to the
hepcidin promoter (Pinto et al. 2008). However, in vivo studies indicate that the down-
regulation of hepcidin is triggered by erythropoietic activity and the increased iron demand
by erythroid precursor cells, rather than directly by EPO (Vokurka et al. 2006). EPO has
also been shown to modulate iron homeostasis by inducing TFR1 expression, and
increasing iron uptake and subsequently haem synthesis in erythroid progenitor cells
(Weiss et al. 1997), and by inhibiting pro-inflammatory immune pathways known to affect
hepcidin and ferritin expression (Nairz et al. 2011). Mechanisms by which erythroid cells in
the bone marrow induce hepcidin suppression are proposed to involve growth-
differentiating factor 15 (GDF15) and twisted gastrulation-1 (TWSG1). GDF15 is a member
of the transforming growth factor (TGF) β superfamily and is predominantly expressed by
erythroid precursors and serum levels of GDF15 are increased in patients with β-
thalassaemia (Tanno et al. 2007), congenital dyserythropoietic anaemias (Casanovas et
al. 2011) and sideroblastic anaemias (Ramirez et al. 2009). Similarly, TWSG1 is also
produced by erythroid precursors and has been shown to increase in thalassaemia and to
suppress hepcidin expression in vitro, by indirectly inhibiting the BMP/SMAD
transcriptional activation of hepcidin (Tanno et al. 2009).
1.7.4 Inflammatory Hepcidin signalling
Inflammatory cytokines and cell stress signals can also modulate hepcidin expression. The
inflammatory up-regulation of hepcidin is considered a mechanism by which the innate
immune system can deprive rapidly growing pathogens of iron. IL-6 has been shown to
promote the phosphorylation of signal transducer and activator of transcription (STAT) 3,
26
which then translocates to the nucleus and upon binding to a proximal promoter element
induces hepcidin transcription (Figure 1.3; (Pietrangelo et al. 2007). In addition, mice
injected with bacterial lipopolysaccharide (LPS) demonstrate increased hepatic hepcidin
expression. This may occur directly via LPS binding to TLR4 (toll-like receptor 4) and
inducing hepcidin expression, or indirectly via increased IL-6 binding to its receptor and
activating the STAT3 pathway in response to LPS binding to TLR4 (De Domenico et al.
2010). IL-1β is also a potent inducer of hepcidin expression utilising both C/EBPα and
BMP/SMAD signalling pathways to induce expression (Matak et al. 2009). In contrast,
tumour necrosis factor (TNF) has been shown to downregulate hepcidin expression in two
ways. Firstly by binding to a HJV response element and inducing its downregulation
(Constante et al. 2007; Salama et al. 2012) and secondly by direct inhibition of SMAD1
protein (Shanmugam et al. 2012). However, previous studies have demonstrated that both
the SMAD1/5/8 and STAT3 pathways are linked by the protein complex Activin B, a TGF-β
protein superfamily and structurally similar to BMP (Kingsley 1994). Activin B has been
shown to induce SMAD1/5/8 phosphorylation in combination with IL-6/STAT3 signalling,
resulting in a marked increase in hepcidin expression (Besson-Fournier et al. 2012). The
induction of hepcidin transcription by endoplasmic reticulum (ER) stress has been
demonstrated to involve the transcription factors cyclic AMP response element-binding
protein (CREB) H and/or the stress-inducible C/EBP homologous protein (CHOP), with
CHOP inducing hepcidin expression through the modulation of C/EBPα activity (Oliveira et
al. 2009). In contrast, oxidative stress has been shown to decrease hepcidin expression,
with increased reactive oxygen species (ROS) promoting the hypoacetylation of histones
leading to decreased binding of C/EBPα and STAT3 to the hepcidin promoter (Choi et al.
2007).
27
1.8 Cellular iron homeostasis Whilst the central role of hepcidin in the regulation of systemic iron homeostasis is well
established, distinct mechanisms that regulate cellular iron homeostasis have also been
identified, allowing for the safe transport and utilisation of iron. Recent studies suggest that
cellular and systemic iron regulatory mechanisms interact with each other to control iron
homeostasis at various levels within an organism (Hentze et al. 2010).
1.8.1 Transferrin Receptor 2 (TFR2) regulation
Iron homeostasis can also be controlled by the cellular regulation of various iron-related
proteins. The iron transporter and regulator, TFR2, has been shown to be regulated at
both a transcriptional and post-translational level, independent of its role in hepcidin
signalling. The murine TFR2 promoter can be activated by the erythroid/liver-related
transcription factors GATA, C/EBP, and erythroid Kruppel-like factor (EKLF). In contrast,
the GATA-1 cofactor, friend of GATA -1 (FOG-1), essential for erythrocyte maturation, was
show to repress the enhanced promoter activity induced by GATA-1 (Kawabata et al.
2001). Also, as mentioned previously, TFR2 protein can be post-translationally stabilised
by the binding of diferric transferrin (Robb and Wessling-Resnick 2004). The cellular
regulation of TFR2 is likely to have a major effect on systemic iron homeostasis via its
signalling to hepcidin, but only a modest effect on cellular iron transport due to its limited
involvement in this process (Chua et al. 2010).
1.8.2 Zrt- and Irt-like Protein 14 (ZIP14) regulation
The iron and zinc transporter ZIP14, is also regulated at a transcriptional level, with IL-6
inducing activation of the ZIP14 promoter (Liuzzi et al. 2005). ZIP14 has also been shown
to be transcriptionally upregulated by IL-1β, which through nitric oxide (NO) signalling
causes the transcription complex AP-1 to translocate to the nucleus where it may to bind
to two putative AP-1 binding sites in the ZIP14 promoter (Lichten et al. 2009). ZIP14 is
also regulated post-translationally, as it has been demonstrated that the presence of HFE
protein results in decreased ZIP14 protein levels, due to decreased stability of the ZIP14
28
protein rather than decreased mRNA levels (Gao et al. 2008). The cellular upregulation of
ZIP14, may play an important role in iron homeostasis during the inflammatory response
and may be responsible for the anaemia of inflammation.
1.8.3 Iron Regulatory Element (IRE)/Iron Regulatory Protein (IRP) regulation
In contrast to TFR2 and ZIP14, many proteins involved in cellular iron uptake, utilisation,
storage and transport are controlled by post-transcriptional mechanisms involving the
IRE/IRP system. Several key proteins of iron metabolism are encoded by mRNAs
containing one or more IREs in their untranslated regions (UTRs). These evolutionary
conserved hairpin structures are binding sites for the two homologous iron regulatory
proteins, IRP1 and IRP2, which are activated for IRE-binding during cellular iron deficiency
(Wallander et al. 2006). IRE/IRP interactions inhibit translation of the mRNAs encoding H-
and L-ferritin, the haem synthesis enzyme, ALAS2, and Fpn, which contain a single IRE in
their 5’ UTR. In addition, IRE/IRP interactions stabilise TFR1 and DMT1 mRNA, which
contain IREs in their 3’ UTR. In conditions of cellular iron deficiency, the IRE/IRP
homeostatic mechanism allows for increased iron uptake and transport via TFR1 and
DMT1 whilst preventing haem synthesis by ALAS2 (Malicka-Blaszkiewicz and Kubicz
1979) and the storage of iron in ferritin and its efflux via FPN. With increasing iron
concentration, IRPs are prevented from binding to IREs. In IRP1, increased iron levels
leads to the reversible insertion of a cubane 4Fe-4S cluster that converts IRP into a
cytosolic aconitase (Wallander et al. 2006). In contrast, increased iron levels result in IRP2
undergoing iron- and oxygen-dependent degradation following ubiquitination by F-
box/LRR-repeat protein 5 (FBXL5), which senses iron levels via a Fe-O-Fe centre within
its haemerythrin domain (Moroishi et al. 2011). IRPs have also been shown to respond to
iron-independent signals, with both IRP1 and IRP2 induced upon exposure of cells to H2O2
(Hausmann et al. 2011) and NO (Wang et al. 2005), stimulating TFR1 expression and iron
uptake.
29
1.8.4 Ferroportin internalisation by hepcidin
The post-translational regulation of FPN has been shown to occur through the binding of
hepcidin to FPN leading to the phosphorylation, internalisation, ubiquitination, and
eventual degradation of FPN (De Domenico et al. 2007). The binding site for hepcidin has
been identified as an extracellular loop in FPN, of which cysteine residue 236 has been
shown to be essential for hepcidin binding (Fernandes et al. 2009). With the dimeric nature
of FPN (De Domenico et al. 2007), each monomer must bind hepcidin for hepcidin-
mediated internalisation. The decrease in FPN protein expression results in decreased
iron release and iron sequesteration in the enterocyte, hepatocyte and macrophage.
1.9 Hereditary haemochromatosis Hereditary haemochromatosis (HH) is an autosomal recessive disorder in which
abnormally high absorption of dietary iron leads to iron accumulation in the parenchymal
tissues. However in the early stages of the disease, macrophages are spared from iron
loading. Excessive iron accumulation is most often observed after an individual reaches
the age of forty, and is most prominent in the liver, pancreas, pituitary, heart, joints and
skin. Accumulation of iron in these areas may lead to liver fibrosis, cirrhosis and
hepatocellular carcinoma, diabetes mellitus, impotence, cardiac failure, arthritis and skin
hyperpigmentation (Siddique and Kowdley 2012). Phlebotomy to remove excess iron is an
effective treatment during the early stages of the disease. HH is a genetically
heterogenous disorder, classified into four types (Olynyk et al. 2008).
1.9.1 Hereditary haemochromatosis (HH) type 1
Type 1 or classical HH is the most common form of the disease. HH type 1 is an
autosomal recessive disorder caused by two common missense mutations, C282Y and
H63D in the HFE gene on chromosome 6p (Feder et al. 1996). Most patients with HH type
1 are homozygous for a C282Y substitution or heterozygotes for C282Y/H63D mutations.
HH type 1 is one of the most prevalent genetic diseases and occurs at a frequency of 1 in
30
200 individuals of Northern European descent (Olynyk 1999). Genetic factors, as well as
environmental factors such as alcohol consumption may contribute to the degree of iron
overload. Patients with HH type 1 lack the ability to downregulate iron absorption and iron
is absorbed via the duodenum at a high rate, irrespective of the body’s iron stores,
resulting in increased serum transferrin saturation and progressive accumulation of body
iron (Andersen et al. 2004). Patients with HH type 1 initially deposit iron in the
parenchymal tissues, primarily in the hepatocytes of the liver, and subsequently in other
organs such as the pancreas and heart leading to irreversible tissue damage. However,
studies have shown that the macrophages of the spleen are comparatively resistant to
dietary iron loading (Harrison 2003).
To study the in vivo consequences of HFE deletion or mutation, five different gene
disruption have been produced in the mouse: a C282Y knock-in (Levy et al. 1999), a H63D
knock-in (Tomatsu et al. 2003), an exon 2-3 knockout (Bahram et al. 1999), an exon 3
disruption/exon 4 knockout (Levy et al. 1999), and the exon 4 knockout (Zhou et al. 1998)
used in this study (Hfe-/-). HFE knockout mice exhibit decreased hepcidin expression
(Ahmad et al. 2002), elevated transferrin saturation (Zhou et al. 1998), and increased
intestinal iron absorption (Ajioka et al. 2002), and as with human HH Type 1 patients, HFE
knockout mice demonstrate relative sparing of iron accumulation in macrophages (Zhou et
al. 1998).
1.9.2 Hereditary haemochromatosis type 2
Juvenile or HH type 2 is characterised by an early onset of iron overload that leads to
severe organ impairment usually before 30 years of age (Camaschella 2002). The disorder
was first described in 1979 and is a recessive trait that affects both sexes.
Hypogonadotrophic hypogonadism and cardiac involvement are prominent features of the
clinical syndrome. The daily increase in iron absorption and the rate of iron accumulation
are higher in HH type 2 HH than in type 1. The first causative gene identified was HAMP1
31
located on chromosome 19q13.1(Roetto et al. 2003) that encodes a peptide, which plays a
key role in the regulation of iron absorption. HH type 2 caused by mutations in the hepcidin
gene is referred to as Type 2B.
To investigate the effects of hepcidin disruption, a mouse with deletion in almost the entire
coding sequence of the Hamp1 gene was generated, with Hamp1 knockout mice
developing early and severe multi-organ iron overload (Lesbordes-Brion et al. 2006), with
the characteristic sparing of the splenic macrophages.
However, for many years it has been known that the most common cause of Type 2 HH
was linked to chromosome 1q, and in 2002 the causative gene was identified and named
haemojuvelin (HJV). A number of mutations in HJV have been identified. The most
common mutations are missense, frameshift, or the insertion of premature stop codons.
Type 2 HH caused by mutations in the HJV gene is referred to as Type 2A. Patients
suffering from Type 2A HH show the same pathology as Type 2B, with the premature
appearance of iron overload (Camaschella et al. 2002).
The Hjv knockout mouse is a model of HH type 2A, which exhibits decreased hepcidin
expression, increased FPN protein in intestinal enterocytes and macrophages (Huang et
al. 2005), and increased iron deposition in the liver, pancreas and heart (Niederkofler et al.
2005).
1.9.3 Hereditary haemochromatosis type 3
HH type 3 is a rare recessive disorder, which leads to iron overload and severe clinical
complications similar to those reported in HH type 1. HH type 3 is caused by mutations in
TFR2, located on chromosome 7q22 (Kawabata et al. 1999). HH type 3 was originally
characterised in two Sicilian families, where several members were found to be
homozygous for a nonsense mutation (Y250X) in TfR2 (Camaschella et al. 2000). In
32
recent years other mutations in TFR2 have been characterised, spread along the entire
sequence of the gene. The clinical phenotype of HH type 3 is similar to HH type 1 with
increased plasma iron and transferrin saturation and parenchymal iron overload.
To investigate HH type 3, two mouse models have been generated. Firstly, as used in the
current study (Tfr2mut), a mouse with a Y245X mutation in Tfr2 (Fleming et al. 2002), a
murine orthologue of the human Y250X mutation, and secondly, a Tfr2 knockout mouse
(Tfr2-/-) (Wallace et al. 2005). Both Tfr2mut and Tfr2-/- mice exhibit inappropriately low
hepcidin levels and hepatic iron overload (Wallace et al. 2005; Drake et al. 2007). In
addition, Tfr2mut mice have been shown to have increased iron absorption, elevated levels
of iron transport genes in the duodenum, and increased liver iron uptake (Drake et al.
2007).
1.9.4 Hereditary haemochromatosis Type 4
HH type 4 is also referred to as FPN disease. Type 4 HH is due to heterozygous mutations
in the FPN gene on chromosome 2q32, which causes mutations in the FPN protein, which
in turn mediates cellular iron export. Type 4 HH differs from other forms of HH for several
reasons. Firstly, it is inherited as an autosomal dominant trait and secondly, patients with
Type 4 HH have high serum ferritin levels but low to normal transferrin saturation. Thirdly,
particularly in young patients there is iron deposition in the reticuloendothelial cells rather
than hepatocytes as found in other type of HH. Patients with Type 4 HH also exhibit
reduced tolerance for phlebotomy and have mild iron-deficient anaemia (Pietrangelo
2004). Of the two forms of HH Type 4, Type 4A is caused by mutations within ferroportin
itself, resulting in an inability to export iron from the cell. Whilst Type 4B is caused by
mutations in the hepcidin binding site of ferroportin, resulting in hepcidin being unable to
degrade ferroportin and as a consequence leading to excessive iron release (Pietrangelo
2004).
33
Currently, there exists only one murine model of HH type 4 and deletion of ferroportin is
embryonically lethal (Donovan et al. 2005). The flatiron mouse, which has the H23R
missense mutation in FPN, exhibits defects in the localisation and export of iron by FPN
(Zohn et al. 2007), resulting in the iron loading of Kupffer cells, high serum ferritin levels,
and low transferrin saturations, similar to patients with HH type 4A.
1.10 Pathogenesis of HH In HH types 1, 2A and 3, one or more components of the plasma iron-sensing machinery
fail, and adequate levels of hepcidin are not produced in response to increased levels of
iron, resulting in increased intestinal and macrophage iron release. When a functional form
of HAMP is expressed at appropriate levels and HFE, TFR2 and HJV are functional, the
amount of iron released into the blood is appropriate for cellular requirements. Disruption
of HFE or TFR2 in HH type 1 and 3 increases the amount of iron that enters the
bloodstream. However, BMP/SMAD signalling is still sufficient to enable some expression
of hepcidin. Therefore, plasma iron loading will proceed at a slower rate and the build up of
iron in parenchymal tissues will be more gradual. The phenotype of iron overload
associated with loss of HJV (HH Type 2B), which is required for hepcidin signalling, is
more severe and similar to that associated with loss of hepcidin itself (HH Type 2A). When
hepcidin expression is normal, mutations in FPN, either in domains that interact with
hepcidin or those that allow FPN to be internalised following hepcidin binding, can result in
a insensitivity to hepcidin and HH type 4.
1.11 Iron induced liver injury Studies have shown that when hepatic iron concentration exceeds 60 µmol/g, hepatic
stellate cells begin to exhibit early signs of activation, an integral event in the initiation of
hepatic fibrosis. As hepatic iron levels increase further, the risk of significant liver fibrosis
34
and ultimately cirrhosis increases (Ramm et al. 1997). Although the exact mechanisms of
liver injury induced by iron overload have not yet been fully elucidated, it is thought that the
accumulation of excess iron-catalysed ROS plays a significant role (Sochaski et al. 2002).
1.11.1 Generation of reactive oxygen species
In almost all cases of oxidative stress the initial reactive oxygen intermediate is the
superoxide radical (O2
•-) which is rapidly converted to hydrogen peroxide (H2O2) by the
action of superoxide dismutases (SODs). Neither O2
•- nor H2O2 are strong oxidising agents
and can usually only interact directly with iron and iron-containing molecules. However,
when redox-active iron is available, the Fenton and Haber-Weiss reactions take place,
where O2
•- reduces Fe3+ to Fe2+, which then reacts with H2O2, resulting in the reactive and
highly damaging hydroxyl radical (OH•)(Aruoma et al. 1991). In the presence of the radical
NO. (produced by nitric oxide synthase) and elevated O2
•-, the peroxynitrite radical (ONOO-
) is formed, which can then react with iron to produce the reactive NO2
+ species (Videla et
al. 2003). Both OH• and NO2
+ are highly reactive and central to the process of lipid
peroxidation and damage to DNA and cellular protein.
1.11.2 Lipid peroxidation
It is suggested that the OH• radical damages the polyunsaturated fatty acids within lipid
membranes resulting in carbon-centred lipid radicals. The intramolecular rearrangement of
the double bonds within these radicals results in the formation of conjugated dienes, which
in the presence of oxygen, form lipid peroxyl radicals (Halliwell and Gutteridge 1984). Lipid
peroxyl radicals can auto-catalyse by reacting with more fatty acids and by forming lipid
hydroperoxide, which is susceptible to cleavage by ferrous and ferric iron molecules, then
decomposing to alkoxyl and peroxyl free radicals, respectively. Lipid hydroperoxides
undergo intramolecular cyclisation and decomposition to generate thiobarbituric acid
(TBA)-reactants and breakdown by-products malondialdehyde (MDA), 4-hydroxynonenal
(4-HNE), ketones, alcohol, ethane and pentane (Bacon and Britton 1990).
35
1.11.3 Lysosmal fragility
In patients with iron overload, the accumulation of iron within lysosomes is commonly
observed (Iancu and Shiloh 1994) and is believed to result from the receptor-mediated
uptake of transferrin (Iacopetta et al. 1983) and ferritin-bound iron (Britton et al. 1994).
Sequestration of iron within the lysosome is believed to be a protective mechanism,
removing iron from redox-sensitive sites and providing a mechanism of excretion via the
biliary system (Britton et al. 1994). Peroxidation of lipids is believed to play a major role in
lysosomal fragility with both ferritin and haemosiderin shown to induce lipid peroxidation in
vitro (O'Connell et al. 1985).
1.11.4 Mitochondrial damage
Lipid peroxidation can also affect the membranes of mitochondria. Chronic iron overload in
vivo in rats (Bacon et al. 1993) and in isolated rat mitochondria (Bacon et al. 1986) has
been shown to have an inhibitory effect on the mitochondrial electron transport chain.
Studies have also shown that lysosomal enzymes can induce mitochondrial oxidant
production and cytochrome c release (Zhao et al. 2003).
1.11.5 DNA damage
Britton and colleagues (Britton et al. 2002; Fleming et al. 2002) have shown that excess
iron can induce DNA damage in both animal models of iron overload and in hepatocytes in
vitro. It has also been demonstrated in haemochromatotic liver that there are increased
levels of etheno-DNA adducts derived from the interaction of lipid peroxidation products
and DNA (Nair et al. 1998), which are associated with the increased frequency of
mutations in the tumour suppressor gene p53 (Hussain et al. 2000). During chronic iron
overload lipid peroxidation produces DNA-reactive aldehydes, which can deregulate
cellular homeostasis and induce malignancy (Nair et al. 1995).
36
1.11.6 Oxidative stress
Previous studies have demonstrated increased hepatic levels of antioxidants, such as
glutathione (Reardon and Allen 2009; Mizukami et al. 2010), and decreased levels of non-
enzymatic antioxidants such as ascorbate, β-carotene and vitamins E and A in iron
overload conditions (Livrea et al. 1996). It has also been demonstrated that iron induced
oxidative stress can initiate apoptosis and necrosis, promoting the synthesis and release
of proinflammatory and fibrogenic factors that alter Kupffer cell (Lin et al. 1997; Xiong et al.
2003) and hepatocyte functions, triggering the activation of hepatic stellate cells and
fibrogenesis (Parola and Robino 2001).
1.11.7 Inflammatory cytokines
Studies have shown that iron overload results in mild hepatic inflammation in response to
apoptotic and necrotic hepatocytes (Deugnier et al. 1992). Inflammatory cells produce a
variety of cytokines involved in the hepatic response to injury. Among these, the
proinflammatory molecule TNF-α , and the anti-inflammatory cytokine IL-10, have emerged
as key factors in iron-induced liver disease (Ramm and Ruddell 2010).
1.12 Present study HH is a primary genetic disorder of iron homeostasis, which results in the hyperabsorption
of dietary iron, leading to iron accumulation in tissues of the body and iron-induced injury
and organ dysfunction (Pietrangelo 2006). Although the aberrant absorption of iron occurs
in the duodenum, mouse models have demonstrated that in most types of HH, the primary
cause of the disorder is the hepatocyte (Verga Falzacappa et al. 2007). It is well
established that hepatocytes take up iron from TBI and NTBI. TBI is taken up by
hepatocytes by TFR1 with high-affinity and to a lesser degree by TFR2 with low-affinity
(Trinder et al. 1996; Chua et al. 2008). NTBI is potentially very toxic as it can generate
ROS. In HH, NTBI is found mainly in the form of iron citrate (Grootveld et al. 1989). In iron
37
overload diseases, such as HH, NTBI uptake becomes important as transferrin saturation
reaches 100% and co-exists with iron citrate in the serum of untreated patients. It is likely
to be an important source of iron accumulation by the liver in iron overload.
Aims
The general aim of this project is to characterise the role of HFE and TFR2 in iron
transport and the regulation of iron metabolism as well as the mechanisms of iron-induced
liver injury caused when these proteins are impaired in HH. The study will utilise the Hfe
knockout (Hfe-/-), Tfr2 Y245X mutant (Tfr2mut) and double mutant (Hfe-/-xTfr2mut) mouse
models of HH.
Hypothesis 1:
Disruption of HFE and TFR2 in combination will result in a form of iron overload that is
more severe than disruption of HFE or TFR2 alone. HFE and TFR2 act in separate as well
as the same signalling pathways as mutations in both genes have an additive inhibitory
effect on hepcidin synthesis causing a more severe iron loading.
Aim 1:
To determine if disruption of HFE and TFR2 in combination results in a form of iron
overload that is more severe than disruption of HFE or TFR2 alone. This aim will be
addressed by cross-breeding Hfe-/- and Tfr2mut mice to produce a Hfe-/-xTfr2mut mouse. The
phenotype including physical characteristics, haematology, iron status (liver iron, plasma
iron and NTBI levels) and expression of key iron metabolism genes will be examined in the
Hfe-/-xTfr2mut mouse and compared with the levels in Hfe-/-,Tfr2mut, wild-type animals (WT)
and dietary iron-supplemented WT mice (WT+Fe).
38
Hypothesis 2:
TFR2 and HFE play an important role in sensing body iron levels, and control iron
metabolism by regulating hepcidin expression, which is impaired in Hfe-/-, Tfr2mut, and Hfe-/-
xTfr2mut mice, leading to increased plasma iron levels and iron deposition in the liver. The
excess iron is taken up by the hepatocyte in the form of NTBI.
Aim 2:
To characterise the mechanisms of hepatic NTBI uptake in Hfe-/-, Tfr2mut, and Hfe-/-xTfr2mut
mice.
This aim will be addressed by measuring plasma NTBI clearance and tissue uptake in vivo
Hfe-/-, Tfr2mut, and Hfe-/-xTfr2mut mice. Tissue NTBI uptake will be correlated with plasma
NTBI levels and tissue iron content, measured by Inductively coupled plasma atomic
emission spectroscopy (ICP-AES).
Hypothesis 3:
In HH mouse models, increasing hepatic iron deposition leads to iron-induced injury, which
recapitulates the injury seen in human HH.
Aim 3:
To examine the iron-induced hepatic pathology associated in the murine models of HH.
This aim will be addressed using Hfe-/-, Tfr2mut, and Hfe-/-xTfr2mut mouse models of HH.
Measurement of iron parameters will be correlated with expression of iron metabolism and
liver injury genes measured by reverse transcription polymerase chain reaction (PCR). In
conjunction, histological, biochemical, and immunofluorescent techniques will be used to
assess lipid peroxidation, collagen deposition and inflammation and the degree of hepatic
injury in the murine models of HH.
39
Hypothesis 4:
Inflammation will increase hepatic NTBI transport in Hfe-/-, Tfr2mut, and Hfe-/-xTfr2mut mouse
models by activation of the IL-6 cytokine pathway, resulting in increased levels of
inflammation-regulated iron transporters and increased iron loading of the liver.
Aim 4:
To investigate the effect of inflammation on the regulation of iron metabolism and iron
transporters in mouse models of HH.
This aim will be addressed using Hfe-/-, Tfr2mut, and Hfe-/-xTfr2mut mice treated with
lipopolysaccharide (LPS) to stimulate IL-6 production. Iron status levels (plasma iron,
transferrin saturation and NTBI levels) will be correlated with iron metabolism gene
expression measured by reverse transcription-PCR.
40
Chapter 2
Materials and Methods
41
Materials
All materials used in this investigation are listed with their supplier below. All reagents and
chemicals are of analytical grade unless specified.
2.1.1 Tissue collection
Material Supplier
Anaesthetics (Ketamine; Xylazine) Troy Laboratories
Heparin Pharmacia and Upjohn
Mouse chow (70 mg or 200mg iron/kg) Specialty Feeds
2.1.2 Experimental procedures
Material Supplier
Acetic acid, glacial BDH
Bathophenanthroline disulfonic acid Sigma
BCA Protein Assay Kit Pierce
Centricon ultrafilters Millipore
HCl BDH
Iron (III) chloride Sigma
Iron (II) sulphate.7H20 BDH
Microfuge tubes Quantum Scientific
MOPS Sigma
Nitriloacetic acid Sigma
Pipette tips Greiner
Serological pipettes Sarstedt
Sodium bicarbonate BDH
Syringes BD Sciences
Thioglycollic acid Sigma
Trisodium Citrate BDH
Triton X-100 BDH
42
2.1.3 Molecular biology
Material Supplier
Agarose Promega
Superscript III Invitrogen
1-Bromo-3-chloropropane Sigma
DNA freeTM kit Ambion
Ethanol Sigma
Isopropanol Sigma
Microfuge tubes (DNAse/RNAse free) Quantum scientific
dNTP Invitrogen
Nuclease free water Invitrogen
oligoDT Invitrogen
Pipette tips (DNAse/RNAse free) Axygen/Fisherbiotec
SYBR Green Master Mix Roche
Primers Geneworks
Invitrogen
Tri-reageent Ambion
RNAsin Promega
2.1.4 Protein extraction and Western blotting
Material Supplier
Complete Mini tablets Roche
PhosphoSTOP Roche
Tris-HCl Sigma
SDS Biorad
Glycerol BDH
Β-Mercaptoethanol Sigma
Bromophenol Blue Sigma
MagicMark XP Invitrogen
Novex Sharp Protein Standard Invitrogen
NuPage Bis/Tris 4-12% gel Invitrogen
MOPS SDS running buffer Invitrogen
NuPAGE transfer buffer Invitrogen
43
Methanol
Nitrocellulose Membrane Fisherbiotec
Antibodies
Rabbit anti-human ferritin DAKO
Goat polyclonal actin Santa Cruz
Donkey anti-goat IgG horseradish peroxidase Santa Cruz
Goat anti-mouse IgG horseradish peroxidase Santa Cruz
Goat anti-rabbit IgG horseradish peroxidase Santa Cruz
Coomassie brilliant blue R-250 Sigma
Western Lightening Detection Perkin-Elmer
PBS ThermoElectron
Novablot electrode paper Amersham
Tween 20 BDH
2.1.5 Equipment
2.1.5.1 Balances
All analytical and biochemical reagents were measured using either an A&D ER-120A or
FX-2000 balances (A&D Mercury Pty Ltd, SA, Australia).
2.1.5.2 Centrifugation
Eppendorf microfuges were used for molecular biology techniques.
2.1.5.2 Imaging system
Images of RNA gels and immunoblots were captured using a VersaDoc imaging system
(Bio-Rad, NSW, Australia).
2.1.5.3 Microscope
A Nikon Eclipse TE-2000U phase contrast microscope (Coherent Scientific, SA, Australia)
and a CK-2 Olympus inverted microscope (Olympus Australia Pty Ltd, Mt Waverley, NSW,
Australia) was used to observe liver pathology.
44
2.1.5.4 Peristaltic pump
A Gilson Minipuls 3 pump (John Morris Scientific, Willoughby, NSW, Australia) was used
for tissue perfusion.
2.1.5.5 pH measurement
A Corning 220 pH meter (Crown Scientific Pty Ltd, Moorebank, NSW, Australia) was used
for all pH measurements. Radiometer buffer solutions were used to calibrate the pH meter.
2.1.5.6 Pipettes
Volumes over 10 mL were measured using a Drummond pipette aid (Drummond Sci Co,
Broomall, PA, USA) and graduated serological pipettes or measuring cylinders. Gilson
(John Morris Scientific, WA, Australia) or Eppendorf (Eppendorf, NSW, Australia)
micropipettors were used to measure volumes of 1 mL or less. A Genex-Beta (Unimed
Australia Pty Ltd, Jandakot, WA, Australia) multichannel variable volume pipette was used
in bicinchoninic acid (BCA) protein assay.
2.1.5.6 Powerpacks
An EPS-301 electrophoresis power supply unit (Amersham Pharmacia Biotech, Castle Hill,
NSW, Australia) was used in conjuction with a semi-dry electroblotter to transfer proteins
onto nitrocellulose membranes. Agarose gels were run in Bio-Rad gel tanks using a Power
Pac 300 power supply (Bio-Rad, NSW, Australia).
2.1.5.7 Radioactivity measurements
Iron-59 (59Fe) samples were counted for 10 min on a Wallac 1480 WizardTM γ-counter
(Perkin Elmer, VIC, Australia).
2.1.5.8 Spectrophotometry
Plasma iron and NTBI concentrations were measured on a Beckman-Coulter DU-640
spectrophotometer (Beckman Coulter Pty Ltd, Gladesville, NSW, Australia). BCA protein
assay measurements were performed spectrophotometrically on a BMG Fluoristar Optima
microplate reader (BMG Labtechnologies Pty Ltd, Mt Eliza, VIC, Australia) and RNA was
quantitated by measurement of absorbance at 260 and 280 nm using a Nanodrop 1000
(Thermo Scientific).
45
2.1.5.9 Real-time PCR
Real-time PCR assays were performed on a Rotor-Gene (QTM or R-G3000TM; Qiagen,
NSW, Australia).
2.1.5.10 Thermocycler
cDNA was amplified on a PTC-100 thermocycler (MJ Research Inc, San Francisco, CA,
USA).
2.1.5.11 Western blot transfer apparatus
Protein samples were electroblotted onto nitrocellulose membranes using the PantherTM
Semi-dry Hep-1 Electroblotter (Owl, Portsmouth, NH, USA).
2.1.6 Location of suppliers
Amersham Pharmacia Biotech, SA, Australia
Axygen Scientific Inc, Mt Martha, VIC, Australia
BDH AnalaR, Kilsyth, VIC, Australia
BD Sciences, Sydney, NSW Australia
Bio-Rad, Regents Park, NSW, Australia
Boehringer Mannheim, Castle Hill, NSW, Australia
DAKO, Glostrup, Denmark
Eppendorf, Clayton, VIC, Australia
Fisons Scientific Equipments, Loughborough, England, UK
GeneWorks, Thebarton, NSW, Australia
GibcoBRL, Invitrogen, Mount Waverley, VIC, Australia
Glen Forrest Stockfeeders, WA, Australia
Greiner Labortechnik Ltd, Kremsmünster, Germany
Invitrogen, Mount Waverley, VIC, Australia
Millipore, North Ryde, NSW, Australia
Pharmacia and Upjohn, WA, Australia
Pierce, Rockford, IL, USA
Promega, Annandale, NSW, Australia
QSP, Quantum Scientific, Milton, QLD, Australia
Santa Cruz Biotechnology; Santa Cruz, CA, USA
Sarstedt AG & Co, Technology Park, SA, Australia
46
Sigma Chemical Co, St Louis, MO, USA
ThermoElectron Co, Melbourne, VIC, Australia
Worthington Biochem Corp, Freehold, NJ, USA
Zymed Laboratories Inc, San Francisco, CA, USA
General methods
2.2.1 Animals
Hfe-/- mice were generated by disruption of the Hfe gene using homologous recombination,
as described by Zhou et al (Zhou et al. 1998). Tfr2mut mice were generated using targeted
mutagenesis to introduce a premature stop codon (C735G) into the murine Tfr2 coding
sequence. The resulting Y245X mutation is orthologous to the Y250X mutation identified in
some patients with HH type 3 (Fleming et al. 2002). Hfe-/- and Tfr2mut mice were
backcrossed for ten generations on to an AKR background at the Animal Resource Centre
(Murdoch, WA, Australia). Hfe-/- and Tfr2mut mice were then crossed to generate Hfe-/-
xTfr2mut double mutant mice. Female Hfe-/-, Tfr2mut, Hfe-/-xTfr2mut and wild-type mice were
fed standard mouse chow (100 mg iron/kg diet; Specialty Feeds, Glen Forrest, WA,
Australia) ad libitum from four weeks of age. An additional group of wild-type mice were
fed an iron-supplemented diet (20 g carbonyl iron/kg diet; Specialty Feeds) for three weeks
from 8-10 weeks of age. Following overnight fasting, blood was collected by cardiac
puncture and organs were perfused in situ with isotonic saline. Tissues were collected and
snap frozen in liquid nitrogen, OCT or fixed in formalin. This study was approved by The
University of Western Australia Animal Ethics Committee.
47
2.2.2 ICP-AES
1 mL of liver homogenate (whole liver homogenised with 1 mL saline), or whole kidney,
pancreas and heart from HH (Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut), non-iron loaded and iron-
loaded WT mice were digested in concentrated 1 mL 12M nitric acid and 1 mL 35%
hydrogen peroxide at 110°C, until volume was halved. The digestion was repeated three
times before samples were made up to 10 mL with Milli-Q water. Measurement of tissue
metals content was performed by Inductively Coupled Plasma Atomic Emission
Spectroscopy using a Varian (Vista AX) ICP-AES CCD Simultaneous, according to AS
3641.2-1999 (Standards Australia International, Strathfield) by the Marine and Freshwater
Research Laboratory (Murdoch University, Perth, WA, Australia).
2.2.3 Plasma Iron Assay
Plasma iron was measured in all samples according to the method described by Fielding
(Fielding 1980). Samples were removed from -80ºC storage and placed on ice until used.
An iron standard solution was prepared from a 20 mM iron stock solution of ferric chloride
dissolved in 1 M HCL. The standard was then diluted 1:4 with Milli-Q water to give a
solution of 280 µL/mL iron in 250 mM HCl, this was then further diluted 1:70 with Milli-Q
water to give a final concentration of 4 µg/mL iron in 7 mM HCl. Dilutions of the stock
solution in Milli-Q water were made to give concentrations ranging from 0-4 µg/mL iron.
50 µL of the plasma or standard were transferred to iron free tubes and an equal volume of
protein precipitate solution consisting of 50% TCA, TGA, HCl (12M) in Milli-Q water was
added to each tube. Samples and standards were mixed thoroughly by vortexing for 30
seconds and incubated at room temperature for 15 min. The samples and standards were
then centrifuged at 14,000 rpm for 15 min. 50 µL of the supernatant was transferred to new
tubes and 50 µL of chromogen solution consisting of sodium acetate, BPS, and Milli-Q
48
water was added. The samples and standards were mixed briefly and allowed to stand at
room temperature for 20 min. Absorbance of each sample and standard was measured
using the Beckman DU 640 spectrophotometer at a wavelength of 535 nm, using Milli-Q
water as a blank. Plasma iron concentrations were then calculated using the standard
curve.
2.2.4 Total Iron Binding Capacity (TIBC)
TIBC was determined in all plasma samples. 50 µL of plasma was transferred to an iron
free tube and 50 µL of saturating iron solution consisting of stock ferric chloride solution
(20 mM) and Milli-Q water was added slowly, to saturate the transferrin with iron. Samples
were mixed thoroughly by vortexing and allowed to stand at room temperature for 15 min.
The samples were centrifuged briefly and 7.5 mg of light magnesium carbonate added to
precipitate any unbound iron. Samples were then mixed gently and placed on a rotating
turntable for 60 min. The samples were then centrifuged at 14,000 rpm for 10 min to
precipitate the magnesium carbonate and the supernatant was transferred to a new tube
and centrifuged at 14, 000 rpm for 7 min. The iron content in 50 µL of the supernatant was
then assayed by the method used in the Plasma Iron Assay.
TIBC concentrations were calculated using the same standard curve generated in the
Plasma iron assay. The iron concentration (µg/mL) of the TIBC samples must be multiplied
by a factor of 2 to account for the 1:2 dilution of the plasma with iron saturating solution in
the TIBC assay, prior to the Plasma Iron Assay.
The data obtained from both the Plasma Iron Assay and the TIBC assay were used to
determine the level of transferrin saturation in the sample using the following formula:
Transferrin Saturation (%) = [Plasma Iron] X 100
[TIBC] 1
49
Levels of transferrin saturation were then corrected for non-transferrin bound iron (NTBI)
by subtracting the NTBI concentration from the transferrin saturation.
2.2.5 Non-Transferrin Bound Iron (NTBI) Assay
Analysis of NTBI levels in HH and wild-type mice was conducted according to the methods
described by Gosriwatana et al (Gosriwatana 1999).
2.2.5.1 Preparation of Tris-carbanatocobaltate(III) trihydrate
A cold slurry of 2.1 g NaHCO3 and 2.5 mL Milli-Q water was prepared at 4ºC. 1.45 g of
cobalt(II)nitrate.6H20 was dissolved in 2.5 mL Milli-Q water and 0.5 mL of 30% H202 was
added dropwise. Cobalt(II)nitrate solution was added to the NaHCO3 solution and
continuously stirred for 1 h at 4ºC. A green precipitate developed and this was separated
by filtration and the precipitate was washed 3 times with 10 mL cold Milli-Q water. After
washing, 1 mL of 100% ethanol was added drop-wise to the precipitate. After all the liquid
was filtered, the precipitate was transferred to a clean filter paper and allowed to dry for 1
h at room temperature, and then for another 1 h at 37ºC. The dry precipitate was removed
from the filter paper and placed in a clean bottle with 10 mL 1M NaHCO3 and stirred for 2 h
at room temperature. The solution was filtered again and the filtrate allowed to stand at
4ºC for 2 days before use. Prior to use the solution was diluted 1:3 with additional NaHCO3
solution.
Frozen plasma samples were removed from -80ºC storage and placed in a water bath at
37ºC for 10 min and the thawed samples were then briefly centrifuged at 13,000 rpm for 30
seconds. 80 µL of plasma was transferred to a clean tube and 17.8 µL of the Tris-
carbanatocobaltate solution was added to each sample and incubated for 60 min in a 37ºC
water bath to saturate all unoccupied iron binding sites with cobalt. 10.85 µL of 800 mM
NTA was added to each sample and incubated at room temperature for 30 min to chelate
50
all non-transferrin bound iron. The Amicon Microcon-30 Centrifugal Filter Devices with a
30,000 molecular weight cutoff were washed with 500 µL of Milli-Q water and then
centrifuged at 14,000 rpm for 12 min. The filters were then turned upside down and
centrifuged again at 2000 rpm for 6 min taking care not to dry out the filters. Samples were
briefly centrifuged before being transferred to the filters and centrifuged for 12 min at 4,000
rpm to separate transferrin bound iron from non-transferrin bound iron. A standard curve
was produced ranging from 0-10 µM iron, using a 50 µM FeCl3 stock solution, made
freshly each time from a 20 mM stock solution.
30 µL of 5mM MOPS buffer at pH 7.4 was added to an equal volume of samples and
standards, followed by 7.5 µL of 120 mM thioglycolic acid. Finally, 7.5 µL of 60 mM BPS
was added and the samples and standard allowed to stand for 30 min at room
temperature. For each sample, a duplicate was prepared containing no BPS and used as
a reagent blank. After 30 min the optical density of the samples was measured using the
Beckman DU 640 spectrophotometer at a wavelength of 535nm, with Milli-Q water used as
a blank.
The line of best fit derived from the standard curve was used to calculate the concentration
of each sample and the concentration of the reagent blank was subtracted from the
concentration in each sample.
2.2.6 RNA extraction
Total RNA was isolated from tissues and gene expression was measured using RT-PCR.
Tissues were homogenised with cold TRI reagent to release RNA from the cells. The
lysates were transferred to 1.5 mL low adhesion tubes and left for 5 min at room
temperature. 1-bromo-3-chloropropane was added to each tube and shaken vigorously for
51
30 seconds. Tubes were then incubated (room temperature, 10 mins) prior to
centrifugation (16100 g, 4oC, 15 mins) to separate RNA (aqueous phase) from DNA and
protein (organic phase). After centrifugation, the aqueous phase was collected and
transferred to fresh 0.6 mL low adhesion tubes. An equal volume of isopropanol was
added to the tubes to precipitate the RNA (room temperature, 10 mins) then centrifuged
(16100 g, 4oC, 15 mins). The supernatant was removed, and the pellet was washed once
with 75% ethanol and centrifuged (16100 g, 4oC, 15 mins). The supernatant was removed
and the pellet was air-dried and dissolved in 15 µL of nuclease free water.
2.2.7 DNase treatment of RNA
RNA samples were treated with DNase to remove any contaminating DNA. 0.1 volume of
10X DNase 1 buffer and 0.5 uL rDNase 1 (2 U/µL) were added to each sample, mixed and
incubated (37oC, 30 mins). Then 0.1 volume DNase inactivating reagent was added to
each sample and incubated (room temperature, 2 mins) with occasional mixing prior to
centrifuging (10,000 g, room temperature, 1.5 min) to pellet the inactivating agent. The
supernatant, containing the RNA, was transferred to fresh low adhesion tubes for storage
at -20oC for short term storage or -80oC for long term storage.
2.2.8 RNA quantification
The concentration and purity of RNA was determined by the measurement of absorbance
at 260 and 280 nm using a Nanodrop 1000 (Thermo Scientific) spectrophotometer using 1
µL of sample. The concentration of RNA was read at A260 where A260 = 1 defined a
concentration of 40 µg/mL. The purity of RNA was determined by the ratio A260/A280 with a
ratio of 1.8-2 representing pure RNA.
52
2.2.9 Gel Electrophoresis
PCR products from samples were run together with 1 Kb Plus Molecular Weight Marker on
a 1.5% agarose gel at 70V for one h. Bands were visualised using a UV transilluminator
and a Kodak digital camera with KDS 120 software.
2.2.10 Reverse transcriptase-Polymerase Chain Reaction (RT-
PCR)
RNA extracted was reverse transcribed to produce complementary DNA (cDNA) and
amplified using polymerase chain reaction. Nuclease free water was added to 1 µg RNA to
a total volume of 5 µL and mixed with RT-Master Mix (Table 2.1) and the tubes were
placed in a PTC-100TM Programmable thermal controller (MJ Research INC.) thermocycler
at 65oC for 5 min to denature RNA. After 5 min, the tubes were placed in a cooling block
for 4 min followed by the addition of the RT-Enzyme Mix (Table 2.1) and incubated at 50oC
for one h. Reverse transcriptase was inactivated by incubating the samples at 70oC for 15
min. After the cycle has completed, the cDNA was stored at 4oC and used within 3 days of
synthesis.
Table 2.1: Reagents used for reverse transcription of RNA.
RT reagents Final Concentration
RT-Master Mix (6.75 µL total volume per reaction)
Oligo dT 25 µg/ mL
dNTPs 0.25 mM
Nuclease free water To volume
RT-Enzyme Mix (3.25 µL total volume per reaction)
RT Reaction buffer 1x
DTT 2.5 mM
RNaseOUTTM 1 U/ µL SuperscriptTM III Reverse
Transcriptase 10 U/ µL
53
Quantification of mRNA transcripts was performed using the Rotorgene RG3000TM
(Corbett Research). Each PCR reaction contained 1 µL of template cDNA and 19 µL of
PCR Master Mix (Table 2.2). The cycling parameters are shown in Table 2.4. During each
cycle, double stranded DNA was denatured, followed by the annealing of primers to the
single stranded DNA template and extension of the primer catalysed by Taq polymerase.
Table 2.2: Reagents in the PCR Master Mix.
PCR reagents Final Concentration
PCR Master Mix (19µL) per reaction
Nuclease free water To volume
FastStart SYBR Green master (Roche)
0.5 X
Primer Mix containing forward and
reverse primer (5µM) (see Table 2.3)
0.25 µM
2.2.11 Primers
Table 2.3: Primer sequences and annealing temperatures.
GENE Primer sequence (5'-3') anneal. temp (°C)
m Beta-actin F-CTGGCACCACACCTTCTA 59
R-GGTGGTGAAGCTGTAGCC
mBMP6 F-ATGGCAGGACTGGATCATTGC 58
R-CCATCACAGTAGTTGGCAGCG
mDcytb F-CATCCTCGCCATCATCTC 56
R-GGCATTGCCTCCATTTAGCTG
mDMT F-TCTATCGCCATCATCCCCACCC 60
R-TCCACAGTCCAGGAAAGACAGACCC
mFPN F-GTCATCCTCTGCGGAATCATCCTGA 58
R-GAGACCCATCCATCTCGGAAAGTGC
mGDF15 F-GAGCTACGGGGTCGCTTC 58
R-K64GGGACCCCAATCTCACCT
mHJV F-TGGTTCTATCAATGGGGGCG 59
R-CACAGTAAAGTTGGGGTCACCG
mHamp1 F-TTGCGATACCAATGCAGAAGA 58
54
R-GATGTGGCTCTAGGCTATGTT
mHFE F-CAGCTGAAACGGCTCCTG 58
R-CGAGTCACTTTCACCAAAGTAGG
mMetallothionein F-CACCAGATCTCGGAATGGAC 60
R-AGGAGCAGCAGCTCTTCTTG
mTransferrin F-CATAACTATGTCACTGCCATTCG 60
R-TCACTGGCGAGTTGTCGAT
mTfR2 F-CCGCTATGGAGACGTGGTT 60
R-TGGCGACACATACTGGGACAG
mZip14A F-TTCCTCAGTGTCTCACTGATTAA 54
R-GGAAAAGGGCGTTAGAGAGC
IL-6 F-GTATGAACAACGATGATGCACTTG 58
R-ATGGTACTCCAGAAGACCAGAGGA
IL-11 F-CTGCACAGATGAGAGACAAATTCC 58
R-GAAGCTGCAAAGATCCCAATG
Il-17a F-GCTCCAGAAGGCCCTCAG 58
R-CTTTCCCTCCGCATTGACA
TGF beta F-TGGAGCAACATGTGGAACTC 60
R-GTCAGCAGCCGGTTACCA
IL-1 alpha F-TTGGTTAAATGACCTGCAACA 60
R-GAGCGCTCACGAACAGTTG
TNF alpha F-CTGTAGCCCACGTCGTAGC 60
R-TTGAGATCCATGCCGTTG
IFN gamma F-ATCTGGAGGAACTGGCAAAA 60
R-TTCAAGACTTCAAAGAGTCTGAGGTA
Id1 F-AACGGCGAGCTCAGTGCCTT 58
R-GAGTCCATCTGGTCCCTCAGTG
Smad7 F'-ACCCCCATCACCTTAGTCG 58
R-GAAAATCCATTGGGTATCTGGA
c-myc F-CCTAGTGCTGCATGAGGAGA 58
R-TCCACAGACACCACATCAATTT
55
Table 2.4: PCR cycling parameters.
Steps Temperature (oC) Time (sec) Number of cycles
Denature 95 600 1
Cycling
– Denaturation 95 15
– Annealing See Table 2.3 20 40
– Extension 72 20
Hold 72 30 1
Melting Curve 72-99 5
Hold 40 30 1
Plasmids containing full-length cDNA or PCR products of the gene of interest were
generated. Standard curves were measured from serial dilutions of known copy numbers
(calculated using known factors: 1 µg of 1000 bp DNA contains 1.52 pmol and 1 mole of
DNA has 6.23 x 1023 molecules) of the plasmids to quantify gene expression in HH and
wild-type mice. The specificity of the PCR product was determined by the melting curve
peak which represents the temperature at which the DNA strands separate for each gene
of interest. This was measured by heating the PCR product and observing the
fluorescence at 1°C increments from 72 to 99°C. mRNA expression of all iron transporters
and regulatory molecules were normalised against β-actin mRNA expression.
2.2.12 Protein extraction
Protein was extracted by homogenisation of tissue samples in 150 mM NaCl, 25 mM Tris-
HCl, pH 7.5 and 0.5% Triton X-100 plus Complete mini protease inhibitors (Roche,
Australia; 1 tablet per 10 mL lysis buffer). The homogenates were left for 1-2 h on ice
before being centrifuged for 15 min at 16000 g at 4°C and the supernatant collected.
Protein concentration of the cytoplasmic extract was then measured using BCA protein
assay.
56
2.2.12.1 Bicinchoninic acid (BCA) protein assay
The Pierce BCA protein assay involves a two-step reaction that combines the reduction of
Cu2+ to Cu+ by protein in an alkaline environment and the selective colourimetric detection
of the Cu+-protein complex with BCA (Smith et al. 1985). Standards were prepared from
known concentrations of bovine serum albumin (BSA) solutions (0-600 µg/mL). Aliquots of
protein and standards (20 µL) were added to the wells of a 96-well tissue culture plate,
followed by the addition of 200 µL of the BCA working reagent (prepared fresh by mixing
49 parts Reagent A to 1 part Reagent B) to each well. The plates were then incubated at
37°C for 1 h and the absorbance measured in a microplate reader.
2.2.13 Statistical analysis
Results are expressed as mean ± SEM where n = 5-11 mice per group, unless otherwise
stated. Differences between groups were analysed using analysis of variance with Tukey’s
multiple comparison post-test or an unpaired Student’s t-test (GraphPad PRISM, La Jolla,
CA, USA). Differences between groups were defined as statistically significant for p < 0.05.
57
Chapter 3
Characterisation of mouse models of hereditary
haemochromatosis
58
3.1 Introduction Iron is an essential trace element with both iron deficiency and iron overload having severe
pathological consequences. For this reason, iron homeostasis is tightly controlled through
the regulation of duodenal iron absorption and iron release from macrophages and other
cell types, with the liver playing a central role in iron storage. A number of the proteins
involved in the regulation of iron homeostasis including HFE, TFR2 and hepcidin are highly
expressed in the liver and are mutated in the various types of HH as outlined in Chapter 1.
A characteristic finding in HH caused by mutations in either HFE or in TFR2 genes is the
decrease in levels of the liver iron regulatory peptide hepcidin compared with livers with
the same degree of liver iron loading caused by secondary iron overload in both humans
and mice (Bridle et al. 2003). This observation suggests that both HFE and TFR2 play a
role in the regulation of hepcidin expression. Recent studies have shown that HFE and
TFR2 are capable of forming a complex (Chen et al. 2007), and evidence suggests this
complex is involved in hepcidin regulation (Gao et al. 2009).
The development of murine HH models has enabled the investigation of the molecular
mechanism responsible for the dysregulation of iron metabolism in HH. The Hfe knockout
model (Hfe-/-) of HH Type 1 (Zhou et al. 1998) and the Tfr2 Y245X knock-in model of HH
Type 3 (Fleming et al. 2002), both have decreased hepcidin levels and increased iron
absorption (Trinder et al. 2002; Drake et al. 2007) and demonstrate how disruption of
either Hfe or Tfr2 genes can result in disturbed iron metabolism akin to that evidenced in
HH patients. Though not classified in its own HH subgroup, there have been reports of
patients with mutations in both HFE and TFR2 that have a form of HH more severe than
either Type 1 or Type 3 (Pietrangelo et al. 2005). The generation of the HFExTFR2 double
mutant model (Hfe-/-xTfr2mut)(Delima et al. 2012) will enable further investigation of the
59
interactions between HFE and TFR2 and their joint roles in iron metabolism and the
pathogenesis of HH.
In this chapter Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut murine models of HH were used as well as
wild-type (WT) mice fed either a control or iron supplemented diet. The aim of this study
was to characterise the phenotypic characteristics of these mice including body and organ
weight, haematological iron parameters, liver metal content as a means of investigating
possible shared transporters of iron, and liver function. Furthermore the effects of
disruption of Hfe and Tfr2 alone or in combination on the expression of duodenal and liver
iron metabolism genes and hepcidin signalling pathways will be examined.
3.2 Methods
3.2.1 Animals Animal models were generated and raised as previously described (Materials and
methods 2.2.1)
3.2.2 Tissue collection Tissues used in this study were collected as stated in Materials and methods 2.2.1.
3.2.3 Haematology Blood was collected by cardiac puncture and placed in Microtainer K2EDTA lined tubes
(BD Medical, NJ, USA). Haemoblobin, haematocrit, red blood cell count (RBC), mean
corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), mean
corpuscular haemoglobin (MCH), red blood cell distribution width (RDW), platelet count
and white blood cell count assays were conducted according to standard diagnostic
methods by PathWest, Fremantle Hospital, Australia.
60
3.2.4 Plasma iron measurement Blood was collected by cardiac puncture according to the method previously described
(Materials and Methods 2.2.1). Plasma iron assays were conducted according to the
methods described in Materials and Methods 2.2.3-5.
3.2.5 Liver function Blood was collected by cardiac puncture and placed in Microtainer serum separating tubes
(BD Medical, NJ, USA). Total protein, albumin, globulin, alanine transaminase and alkaline
phosphatase assays were conducted according to standard diagnostic methods by
PathWest, Fremantle Hospital, Australia.
3.2.6 Hepatic metal measurement Hepatic metal (iron, copper, zinc and manganese) content was measured by ICP-AES as
previously described (Materials and methods 2.2.2)
3.2.7 Gene expression Total RNA from liver tissue was isolated as described in Materials and methods 2.2.6.
Measurement of liver gene expression was performed by RT-PCR according to the
manufacturer’s instructions as outlined in Materials and methods 2.2.10. Real-time PCR
was conducted for liver β-Actin, Bmp6, DcytB, Dmt1, Fpn, Hamp1, Hfe, Id1, Smad7, Tfr1,
Tfr2, Transferrin, Zip14 using the appropriate forward and reverse primers (Table 2.3) on a
Rotorgene 3000 as previously described (Materials and methods 2.2.10-11).
3.2.8 p-Smad 1/5/8 expression Measurement of p-Smad 1/5/8 was performed by Western blotting. Briefly, liver
homogenates (100 µg) were electrophoresed on 4%-12% Novex Bis-Tris gradient gels
(Invitrogen) with a MOPS-SDS buffering system, prior to being transferred onto BioTrace
NT nitrocellulose membrane (Pall, NY, USA) Membranes were blocked in 5% skim milk
powder in TBST (tris-buffered saline and 0.5% Tween 20) for 30 min at room temperature.
Membranes were incubated sequentially in anti-phospho-Smad1/5/8 (1:500, Cell
61
Signalling, Boston, MA,USA), anti-Smad1/5/8 (1:500, Santa Cruz Biotechnology, Santa
Cruz, CA, USA) and anti-β-actin (1:1000, Millipore, Billerica, MA, USA) in PBST, overnight
at 4ºC. Membranes were washed in TBST before incubation in anti-rabbit (Millipore) or
anti-mouse (Millipore) horseradish peroxidase for 4 h at room temperature. After
incubation, membranes were washed in 3 changes of TBST. Following a final wash in
TBST, Western Lightning Chemiluminescent reagent Plus (PerkinElmer, MA, USA) was
applied to membranes and images were captured using a VersaDoc Model 3000 (BioRad,
Hercules, CA, USA) and protein levels were quantified using Quantity One software
(BioRad).
3.2.9 Statistics Results are expressed as mean ± SEM, n=6 and were calculated as previously described
(Materials and methods 2.2.13).
3.3 Results Total body and organ weights in HH mice (Hfe
-/-, Tfr2mut, Hfe
-/-xTfr2mut) and wild-
type (WT) mice (iron-loaded and non-iron-loaded) are shown in Table 3.1. Total
body weight was approximately ten percent lower in Hfe-/-xTfr2
mut, Hfe
-/- and iron-
loaded WT mice compared with their non-iron loaded wild-type counterparts.
Organ weight is expressed relative to total body weight. Liver weight was similar in
Hfe-/-, Tfr2
mut and WT mice and significantly (p<0.05) increased in Hfe-/-xTfr2
mut
compared with Tfr2mut and WT mice. Splenic weight was unchanged in single
mutant mice and iron-loaded mice compared with non-iron-loaded WT mice.
However, splenic weight was 33% higher in Hfe-/-xTfr2
mut than single mutant and
WT mice. The kidney weight was increased in all HH mice compared with WT mice
but was not significantly different between HH mice. There was no variation
observed in heart, pancreas and brain weight between all types of mice.
62
Table 3.1: Body and organ weights of HH and WT mice.
WT WT+Fe Hfe-/- Tfr2
mut Hfe-/-xTfr2mut
Body weight (g)
25.8±0.5 22.9±0.4a 23.9±0.4a 25.5±0.4 23.3±0.5a
Liver (mg/g body weight)
48.3±0.9 48.5±1.3 48.5±1.9 50.1±0.9 53.4±0.7a,b,d
Kidneys (mg/g body weight)
12.4±0.2 12.3±0.2 13.1±0.3a,b 12.8±0.2a,b 12.7±0.2a,b
Spleen (mg/g body weight)
3.1±0.1 3.1±0.1 3.1±0.2 3.2±0.1 3.7±0.2a,b,c,d
Heart (mg/g body weight)
6.0±0.5 5.6±0.2 5.9±0.1 5.6±0.1 5.8±0.1
Pancreas (mg/g body weight)
12.2±0.2 12.3±0.4 12.6±0.5 12.3±0.3 11.9±0.3
Brain (mg/g body weight)
17.7±0.3 18.7±0.3 18.7±0.3 17.7±0.2 18.9±0.6
NOTE. Results are expressed as mean ± SEM (n=5-15). ap<0.05 vs WT; bp<0.05 vs WT+Fe; cp<0.05 vs Hfe-/-; dp<0.05 vs Tfr2mut denote significance between groups.
63
3.3.1 Haematology Both plasma haemoglobin and haematocrit (Hct) levels were significantly (p<0.05)
increased in Tfr2mut and Hfe-/-xTfr2mut mice compared with Hfe-/- and WT mice (Table 3.2).
Hfe-/-xTfr2mut and Tfr2mut mice also displayed approximately a 10% increase in red blood
cells (RBC) when compared with Hfe-/- and non-iron-loaded WT mice. Mean corpuscular
volume (MCV) was not significantly altered in Hfe-/-xTfr2mut, Hfe-/-, iron-loaded WT mice
compared with non-iron-loaded, but was increased in Tfr2mut mice when compared to all
other types of mice. Mean cell haemoglobin concentration (MCHC) was increased in Hfe-/-
xTfr2mut, Hfe-/-, and iron-loaded WT mice compared with non-iron-loaded mice and Tfr2mut
mice. No significant changes in mean cell haemoglobin (MCH), red blood cell distribution
width (RDW), or platelet number were observed (Table 3.2). White cell counts were
unlatered in Hfe-/-xTfr2mut, Hfe-/-, and iron-loaded WT mice compared with non-iron-loaded
mice but were elevated in Tfr2mut mice compared with all other mice (Table 3.2).
64
Table 3.2: Haematological parameters in HH and wild type mice.
WT WT+Fe Hfe-/- Tfr2
mut Hfe-/-xTfr2mut
Haemoglobin (g/L)
137.0±2.5 147.4±3.0a 142.3±2.1 158.1±2.0a,b,c 158.6±2.6a,b,c
Hct 0.45±0.01 0.48±0.01 0.44±0.01 0.52±0.01a,b,c 0.50±0.01a,b,c
RBC (x1012/L)
8.47±0.18 9.33±0.24a 8.56±0.17 9.55±0.11a,c 9.58±0.17a,c
MCV (fL)
52.67±0.21 50.57±0.20a 51.30±0.54 54.45±0.37a,b,c 52.44±0.24b,d
MCHC (g/L)
303.4±4.2 317.0±3.4a 324.3±4.4a 303.6±3.3c 316.9±1.1a,d
MCH (pg)
16.32±0.40 16.10±0.10 16.61±0.19 16.54±0.10 16.58±0.08
RDW 14.32±0.31 13.81±0.21 13.75±0.23 14.24±0.14 14.10±0.23
Platelets (x109/L)
365.2±104.1 317.0±91.7 413.6±58.1 440.6±48.3 237.3±38.1
White cells (x109/L)
3.85±0.41 3.71±0.34 4.10±0.37 5.14±0.29a,b,c 3.47±0.34d
NOTE. Results are expressed as mean ± SEM (n=5-15). ap<0.05 vs WT; bp<0.05 vs WT+Fe; cp<0.05 vs Hfe-/-; dp<0.05 vs Tfr2mut denote significance between groups. RBC, red blood cell count; Hct, haematocrit; MCV, mean corpuscular volume; MCH, mean cell haemoglobin; MCHC, mean cell haemoglobin concentration; RDW, red blood cell distribution width.
65
3.3.2 Plasma Iron parameters Plasma iron concentration and transferrin saturation were higher in Hfe-/-xTfr2mut, Tfr2mut,
Hfe-/- and iron-loaded WT mice compared with non-iron-loaded WT mice (p<0.05; Fig.
3.1A,B). Plasma iron concentration and transferrin saturation were highest in Hfe-/-xTfr2mut
mice (p<0.05, Fig. 3.1A,B). Plasma iron concentration in Tfr2mut mice was increased
compared to Hfe-/- mice (p<0.05). Plasma NTBI concentration was also elevated in all iron-
loaded mice (p<0.05). In Hfe-/-xTfr2mut mice, NTBI levels were 7-fold higher than non-iron-
loaded wild-type mice and more than 2-fold higher than Hfe-/-, Tfr2mut and iron-loaded WT
mice (p<0.001; Fig. 3.1C).
66
Figure 3.1: Plasma iron parameters. Plasma iron concentration (A), transferrin saturation (B) and non-transferrin bound iron (NTBI) concentration (C) were measured in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut.
67
3.3.3 Liver function
Plasma total protein and globulin levels were only increased in Hfe-/-xTfr2mut and Tfr2mut
mice compared with WT mice. Tfr2mut mice had increased levels of plasma albumin when
compared to Hfe-/- and WT mice, whilst no change was observed in Hfe-/-xTfr2mut mice
(Table 3.3). Serum alanine transaminase (ALT) and alkaline phosphatase are markers of
liver injury. ALT levels were increased in all HH mice and iron-loaded WT mice. Hfe-/-
xTfr2mut ALT levels were also significantly (p<0.05) increased in compared with single
mutant HH mice and were more than 3-fold higher than WT mice (Table 3.3). Alkaline
phosphatase levels were unaltered in Hfe-/-xTfr2mut and Tfr2mut mice but were increased in
Hfe-/- and iron-loaded WT mice compared with non-iron-loaded WT mice.
68
Table 3.3: Serum markers of liver function in HH and wild type mice.
WT WT+Fe Hfe-/- Tfr2
mut Hfe
-/-xTfr2mut
Total protein (g/L) 46.83±1.05 45.25±0.45 47.17±1.51 54.27±0.76a,b,c 51.70±0.54a,b,c
Albumin (g/L) 26.83±0.75 26.88±0.40 26.33±1.02 30.09±0.56a,b,c 27.09±0.48d
Globulins (g/L) 20.00±1.00 18.38±0.18 20.83±0.54b 24.18±0.63a,b,c 24.30±0.37a,b,c
Alanine transaminase (U/L)
87.80±7.27 170.75±11.99a 162.40±8.56a 142.00±7.90a 294.00±55.70a,b,c,d
Alkaline Phosphatase (U/L)
102.83±8.17 130.25±5.28a 137.40±2.34a 110.09±2.03b,c 103.91±4.02b,c
NOTE. Results are expressed as mean ± SEM (n=5-15). ap<0.05 vs WT; bp<0.05 vs WT+Fe; cp<0.05 vs Hfe-/-; dp<0.05 vs Tfr2mut denote
significance between groups.
69
3.3.4 Liver metal content Hepatic iron content (HIC) was more 20-fold higher than liver zinc content which was in
turn more than 10-fold higher than liver copper and manganese content (Fig. 3.2). HIC
was higher in Hfe-/-xTfr2mut mice compared with either Hfe-/- or Tfr2mut mice (Fig. 3.2A,
p<0.01) and approximately 5-fold greater than non-iron-loaded WT mice. In Tfr2mut mice
HIC was approximately 20% higher than in Hfe-/- mice which were similar to the iron-
loaded WT and 3-fold higher than non-iron-loaded WT (Fig. 3.2A, p<0.001). Hepatic zinc
concentration was increased only in Hfe-/-xTfr2mut mice (Fig. 3.2B, p<0.01), whilst copper
concentration was increased in both Tfr2mut and Hfe-/-xTfr2mut mice compared with other
types of mice (Fig. 3.2C, p<0.05). Hepatic manganese concentration was only altered in
Hfe-/-xTfr2mut mice, where it was increased by approximately 2-fold compared with WT
mice (Fig. 3.2D, p<0.05).
70
Figure 3.2: Liver metal content Liver metal content was measured by ICP-AES. Iron (A), zinc (B), copper (C), and manganese (D) concentrations were measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut denote significance between groups.
71
3.3.5 Liver expression of iron regulatory genes Hfe expression was undetectable in Hfe-/- and Hfe-/-xTfr2mut mice (p<0.001) while
expression was similar in Tfr2mut and WT mice (Fig 3.3A). Tfr2 mRNA expression in Tfr2mut
and Hfe-/-xTfr2mut mice was decreased by more than 80% compared with non-iron-loaded
WT mice (p<0.001). Tfr2 mRNA expression in Hfe-/- and iron-loaded WT mice was also
lower than non-iron-loaded wild-type mice (p<0.05; Fig 3.3B). Expression of Hamp1 was
decreased to approximately 40% in Hfe-/- and Tfr2mut mice compared with non-iron-loaded
WT mice. In Hfe-/-xTfr2mut mice, Hamp1 expression was almost abolished, being further
reduced to approximately 1% or 3% of that observed in non-iron-loaded wild-type mice
(p<0.01) or Hfe-/- and Tfr2mut mice (p<0.05), respectively. Hamp1 expression, as expected,
was increased in iron-loaded wild-type mice compared with non-iron-loaded wild-type mice
(p<0.05) and HH mice (p<0.001; Fig 3.3C). Bmp6 mRNA expression was similar in Hfe-/-
xTfr2mut, Tfr2mut and Hfe-/- mice and approximately double that of the non-iron-loaded WT
mice. However, Bmp6 expression was greatest in the iron-loaded WT mice which were
almost 60% higher than the Hfe-/-xTfr2mut, Tfr2mut and Hfe-/- and were more than 2-fold
higher than in non-iron-loaded WT mice (p<0.001; Fig 3.3D). Liver expression of Id1
mRNA was similar and decreased in Hfe-/-xTfr2mut, Tfr2mut and Hfe-/- mice but was markedly
upregulated in iron-loaded WT compared with non-iron-loaded WT (p<0.001; Fig 3.3E).
Smad7 expression was 30% lower in Hfe-/-xTfr2mut than in Tfr2mut, Hfe-/- and non-iron-
loaded WT mice which were similar and 3-fold lower than in the iron-loaded WT mice
(p<0.001; Fig 3.3F).
72
Figure 3.3: Liver expression of iron regulatory genes. mRNA expression was determined by real-time qPCR. Hfe (A), Tfr2 (B), Hamp1 (C), Bmp6 (D), and Id1 (D) mRNA expression were measured in WT), WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, p<0.05 vs. WT; b, p<0.05vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut denote significance between groups.
73
3.3.6 Liver expression of SMAD1/5/8 Hepatic phosphorlyation of SMAD1/5/8 protein (p-Smad1/5/8) was reduced by almost 50%
in Hfe-/-xTfr2mut mice compared with non-iron-loaded WT mice (Fig 3.4A+B). pSmad1/5/8
levels were unchanged in Hfe-/- and Tfr2mut mice compared to non-iron loaded WT mice but
was decreased by 30% compared with iron-loaded WT mice. pSmad1/5/8 levels in iron-
loaded wild-type mice were 30% higher than non-iron-loaded WT mice (Fig 4A+B). No
change was detected in total SMAD1/5/8 protein (SMAD1/5/8) in any groups.
74
Figure 3.4: Hepatic p-Smad1/5/8 protein expression. Total liver protein from WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut mice was immunoblotted with antibodies against phospho-Smad1/5/8, and total Smad1/5/8, with actin as a loading control (A). Protein levels were quantified by densitometry and phospho-Smad levels were expressed relative to actin and normalised to WT levels. Results are expressed as mean ± SEM (n=6). a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut denote significance between groups (B).
75
3.3.7 Liver expression of iron transport genes Liver Tfr1 mRNA expression was reduced in all types of HH mice and iron-loaded WT
mice compared with non-loaded WT mice (p<0.05), consistent with liver iron loading.
There was no difference in the level of Tfr1 expression between Hfe-/-xTfr2mut, Hfe-/- and
Tfr2mut mice (Fig 3.5A). Similarly Dmt1 mRNA expression was decreased by almost 50% in
HH and iron-loaded WT mice (p<0.05; Fig. 3.5B). Liver mRNA expression of the
transmembrane metal transporter Zip14 was decreased in all iron-loaded mice. It was
decreased by approximately 50% in Hfe-/-, Tfr2mut and iron-loaded WT mice and was
further decreased in Hfe-/-xTfr2mut mice to approximately 30% of non-iron loaded WT levels
(Fig. 3.5C). Expression of the iron transport protein Tf was increased in HH and iron
loaded WT mice compared with non-iron loaded WT mice. Tf expression was significantly
(p<0.05) greater in HH mice than iron-loaded WT mice (Fig. 3.5D). mRNA levels of the iron
exporter Fpn were unchanged in HH and WT mice (Fig. 3.5E).
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Figure 3.5: Liver expression of iron transporter genes. mRNA expression was determined by real-time qPCR. Tfr1 (A), Dmt1 (B), Zip14A (C), Transferrin (D) and Fpn mRNA expression (E) were measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut denote significance between groups.
77
3.3.8 Duodenal expression of iron transport genes Duodenal Tfr1 expression was unchanged in Tfr2 mutant and Hfe-/-xTfr2mut mice but was
decreased in Hfe-/- mice when compared with WT mice (Fig. 3.6A). Duodenal Dmt1 mRNA
expression was increased by 2-fold in Tfr2mut and Hfe-/-xTfr2mut mice but was unchanged in
Hfe-/- and iron-loaded WT mice compared with non-iron loaded WT mice (Fig. 3.6B).
Expression of the duodenal iron exporter Fpn was significantly (p<0.05) increased in
Tfr2mut and Hfe-/-xTfr2mut mice compared with WT mice but was unchanged in Hfe-/- and
iron-loaded WT mice compared with non-iron loaded WT mice (Fig. 3.6C). Duodenal
expression of DcytB mRNA was increased by more than 10-fold in Tfr2mut and Hfe-/-xTfr2mut
mice and by 2-fold in Hfe-/- mice when compared with WT mice (Fig. 3.6D).
78
Figure 3.6: Duodenal expression of iron transport genes. Gene expression was determined by real-time qPCR. Tfr1 (A), Dmt1 (B), Fpn (C), and DcytB (D) mRNA expression were measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, p<0.05 vs. WT; b, p<0.05vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut denote significance between groups.
79
3.4 Discussion In this study it was shown that Hfe-/-xTfr2mut mice develop a more severe iron loading
phenotype than their Hfe-/- or Tfr2mut single mutant counterparts. Hfe-/-xTfr2mut mice were
found to have decreased body weight and increased liver, kidney and spleen weights
when compared with non-iron-loaded WT mice. Haematological parameters (RBC, Hb,
and Hct), iron status (plasma iron, transferrin saturation and NTBI concentration) and liver
metal content (iron, zinc, copper and manganese) were all increased in Hfe-/-xTfr2mut mice.
Hfe-/-xTfr2mut mice had increased Bmp6 levels consistent with increased HIC but
decreased Hfe and Tfr2 expression lead to ineffective p-Smad signaling and reduced
Hamp1 expression.
The observation that the murine models of HH; Hfe-/-xTfr2mut and Tfr2mut had increased
numbers of RBCs with elevated haemoglobin and haematocrit levels consistent with
several other studies in HH patients (Barton et al. 2000; McLaren et al. 2007) and mice
(Ramos et al. 2011) that have observed haematological changes. It is thought that HH
increases erythropoiesis in two ways; firstly, the down regulation of hepcidin in HH results
in increased iron absorption by the intestine and increased recycling of iron from the
macrophage increasing the bioavailability of iron for erythropoiesis (Zhang et al. 2011).
Secondly, TFR2, is abundantly expressed in the early erythron (Sposi et al. 2000) and it
has been shown that the binding of TFR2 to the erythropoietin receptor (EpoR) is required
for efficient erythropoiesis (Forejtnikova et al. 2010). Other studies have shown that
erythroid progenitor cells from Tfr2 deficient mice are less responsive to erythropoietin
(Epo), resulting in increased serum Epo levels further stimulating erythropoiesis. The
increase in erythropoiesis in Hfe-/-xTfr2mut mice is further supported by the increase in
spleen weight (Table 3.1), as splenomegaly is often associated with increased workload by
the spleen in response to proliferation of the bone marrow (Pozo et al. 2009).
80
Plasma iron concentration and transferrin saturation were increased in Hfe-/- and Tfr2mut
mice in congruence with the characterisation studies of these mice by Fleming et al (Zhou
et al. 1998; Fleming et al. 2002) and were further increased in Hfe-/-xTfr2mut mice (Wallace
et al. 2009; Corradini et al. 2011). HH mice also had increased NTBI levels providing
further evidence that HH is a disease of dysregulated iron absorption, where absorbed iron
exceeds the iron binding capacity of transferrin and the excess iron in present in the serum
as NTBI.
Increased serum total protein, globulin and ALT levels observed in Hfe-/-xTfr2mut mice
suggest that Hfe-/-xTfr2mut mice have liver injury. This is further supported by the
observation of hepatomegaly in the Hfe-/-xTfr2mut mice, which is a common occurrence in
the advanced liver disease caused by HH (Brissot et al. 2000). The effect of excess
hepatic iron deposition on liver injury in HH mice will be described in more detail in
Chapter 5.
In this study it has been shown that Hfe-/-xTfr2mut mice develop a more severe hepatic iron
loading than Hfe-/- and Tfr2mut mice (Fig. 3.1). This observation is in agreement with reports
of a severe form of HH with an early onset akin to that seen in juvenile HH, in probands
with mutations in both HFE and TFR2 (Pietrangelo et al. 2005; Rueda Adel et al. 2011).
Other studies in mice with disruptions in both HFE and TFR2 (Wallace et al. 2009;
Corradini et al. 2011) have shown increases in hepatic iron concentration that are similar
to that observed in the current study. The increase in liver zinc, copper and manganese
content in Hfe-/-xTfr2mut mice suggests that there is a shared transporter for these divalent
metals and iron which is upregulated in the absence of HFE and TFR2, possible
transporters include DMT1 and ZIP14 (Hansen et al. 2009). Further evidence of a shared
transporter for the metals iron, zinc, copper and managense is the study by Chua et al
(Chua et al. 2004) who found that the divalent metals manganese, zinc, and copper
81
inhibited ferric citrate uptake by hepatocytes from both Hfe-/- and wild-type mice. Inhibition
of NTBI uptake has also been demonstrated by zinc and manganese in the perfused rat
liver (Wright et al. 1986), and by zinc in both isolated rat hepatocytes (Baker et al. 1998),
and hepatoma cells (Randell et al. 1994). Increased levels of zinc, copper and manganese
in Hfe-/-xTfr2mut mice may play a role in oxidative stress as copper, zinc and manganese
are important components of the free radical scavenger, superoxide dismutase (McCord
and Fridovich 1988).
The reduction in liver hepcidin (Hamp1) expression in the presence of disruption of either
Hfe or Tfr2 in the liver emphasises the role that HFE and TFR2 play in the regulation of
hepcidin expression and iron homeostasis. Furthermore, the additive effect on iron loading
by the near abrogation of hepcidin in response to the disruption of HFE and TFR2,
suggests they may work in parallel or possibly converging signalling pathways. Other
studies have demonstrated that the iron-dependent regulation of hepcidin is controlled by
HFE and TFR2, and BMP6/SMAD cell signalling pathways (Kautz et al. 2008; Wallace et
al. 2009). It has been shown that HFE can interact with TFR1 and TFR2 to form a complex
that is hypothesised to sense plasma transferrin saturation and modulate hepcidin
synthesis accordingly (Olynyk et al. 2008). However, the exact nature of this mechanism is
yet to be fully elucidated. These findings support previous studies that suggest there may
be cross-talk between HFE/TFR2 and BMP6/SMAD signalling pathways, as disruption of
Hfe in combination with Tfr2 results in decreased levels of pSMAD1/5/8, which leads to a
decrease in hepcidin synthesis.
Duodenal expression of the iron transporters Dmt1 and Fpn was unchanged in Hfe-/- mice
but was significantly (p<0.05) increased in Tfr2mut and Hfe-/-xTfr2mut mice. This result is
consistent with other studies that show Dmt1 and Fpn are unchanged in Hfe-/- mice
(Herrmann et al. 2004) but are both increased in Tfr2mut mice (Drake et al. 2007). Reports
82
of increased levels of duodenal Dmt1 and Fpn mRNA in Hfe-/- (Dupic et al. 2002) and
Tfr2mut mice (Kawabata et al. 2005) are most likely due to genetic differences in the
background stains of the mutant mice. Increased iron absorption has been demonstrated
in Hfe-/- mice (Ajioka et al. 2002) and Tfr2mut mice (Drake et al. 2007) and it is likely to be
similar in Hfe-/-xTfr2mut mice. The enterocytes of the duodenum have been shown to be
relatively iron deficient in HH (Francanzani et al. 1989) due to the excessive export of iron
to the circulation. The increase in Dmt1 expression in the Tfr2mut and Hfe-/-xTfr2mut mice is
likely to be due to an increase in DMT1 transcripts containing an IRE that is increased in
iron deficiency (Hubert and Hentze 2002). This is supported by reports of high duodenal
Dmt1 expression in iron deficient mice (Gunshin et al. 1997). The reason for an increase in
Fpn in Tfr2mut and Hfe-/-xTfr2mut mice is unclear but as mentioned previously, similar
findings have been reported (Drake et al. 2007; Wallace et al. 2009).
Hepatic TfR1 expression was decreased in all HH mice (Fig. 3.3A). This finding was
expected and is supported by previous studies in Hfe-/- (Chua et al. 2008), Tfr2mut (Chua et
al. 2010) and iron-loaded wild-type mice (Chua et al. 2008; Chua et al. 2010). TfR1
expression is inversely regulated by cellular iron levels by the IRE/IRP post-transcriptional
regulation as described in Chapter 1 (section 1.8.3), wherein Tfr1 mRNA undergoes
enhanced degradation under conditions of high HIC found in HH mice (Rouault 2006). The
fact that liver Fpn mRNA levels were unchanged by high HIC in the current study (Fig.
3.3B), is consistent with the findings of previous studies (Chua et al. 2008; Chua et al.
2010); (Constante et al. 2006) and support the theory that FPN regulation occurs by a
post-translational mechanism, with hepcidin binding to FPN protein and inducing its
degradation (Nemeth et al. 2004).
The increased expression of liver transferrin in Hfe-/-xTfr2mut, Hfe-/- and Tfr2mut mice is an
unexpected finding as the hepatic synthesis of transferrin has been shown to be positively
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regulated by iron deficiency, not iron overload. However, proximal region II, adjacent to the
transferrin promoter of transferrin contains a CCAAT sequence capable of binding CCAAT
enhancer-binding proteins (C/EBP) (Theisen et al. 1993). One particular form of C/EBP,
known as C/EBPα has been shown to be positively regulated by increased iron levels
(Harrison-Findik et al. 2007) and may possibly contribute to the iron dependent regulation
of transferrin seen in the HH mice.
Hepatic expression of the zinc and iron transporter Zip14 was decreased in Hfe-/- and
Tfr2mut mice and to a greater extent in Hfe-/-xTfr2mut mice. The decrease in Zip14A
expression with increasing HIC may be due to in the increased hepatic zinc content
evident in Hfe-/-xTfr2mut mice. Zip14 is a member of slc39 transporter family, other
members of the family such as Zip10 are known to possess metal-response elements
which in response to zinc-induced metal-regulatory transcription factors result in the down-
regulation of the target mRNA (Zheng et al. 2008). Zip14 may also be regulated in a
manner similar to Zip4, where zinc excess results in reduced mRNA stability leading to
protein degradation (Mao et al. 2007). It has also been shown that in the absence of HFE,
the stability of ZIP14 protein is increased (Gao et al. 2008) allowing for ZIP14 to contribute
to liver iron and zinc transport in HH despite a decrease in mRNA expression.
The disruption of HFE and TFR2 results in an inability to sense plasma iron levels and
impairment of Bmp/Smad signaling resulting in reduced hepcidin synthesis. With the loss
of hepcidin synthesis, duodenal iron absorption continues unabated and the excess iron
that is responsible for the alterations in phenotype that were evident particularly in Tfr2mut
and Hfe-/-xTfr2mut mice. Increased liver iron and other metal levels in Hfe-/-xTfr2mut mice
result in hepatomegaly with increased erythropoiesis leading to elevations in RBCs and Hb
levels and ultimately splenomegaly (Spivak 2000). The results of this study suggest that
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disruption of HFE and TFR2 whilst primarily causing liver iron overload also induced other
phenotypic changes that should be considered in human HH.
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Chapter 4
Non-transferrin-bound iron transport in hereditary
haemochromatosis
86
The following chapter "In vivo liver non-transferrin-bound iron uptake is upregulated in
murine models of Hereditary Hemochromatosis" has been submitted to American Journal
of Physiology - Gastrointestinal and Liver Physiology on 4th Apr 2013 (GI-00113-2013)
87
4.1 Introduction
Under normal conditions, the plasma iron transport protein, transferrin, is 20-35%
saturated with iron (Morgan 1996). However, in conditions where transferrin approaches
saturation, such as hereditary haemochromatosis and in transfusion-dependent
thalassaemias, excess iron in the body is unable to bind to transferrin and exists in the
circulation as non-transferrin-bound iron (NTBI). NTBI can produce reactive oxygen
species (ROS) in tissues and bodily fluids through catalysis of the Fenton reaction
(Aruoma et al. 1988).
Since the early studies by Hersko et al. (Hershko et al. 1978), NTBI has been found to be
present in several other iron-overload conditions such as sickle cell anaemia (Koren et al.
2010) and bone marrow failure during cancer treatments (Sahlstedt et al. 2009). Though
the presence of NTBI usually correlates with high transferrin saturation, it has also been
found in patients with only partially saturated transferrin (Gutteridge et al. 1985). Increased
NTBI levels have been implicated in abnormal iron deposition in the liver, heart and
endocrine glands (Oudit et al. 2003) and are thought to be a major cause of organ
dysfunction in iron-overload disorders.
Though the presence of NTBI has been known for many years, there is still a poor
understanding of its chemical composition in various iron-overload disorders. NTBI in the
plasma may exist as iron bound to various non-protein ligands such as citrate, acetate,
pyruvates and phosphates (Silva and Hider 2009), though a precise understanding of the
chemical composition of NTBI has yet to be elucidated. However, studies using serum
from haemochromatosis patients, implicates iron-citrate as the predominant form of
circulating NTBI in haemochromatosis (Grootveld et al. 1989).
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The uptake and distribution of NTBI in hereditary haemochromatosis is still poorly
understood. In this condition, excess circulating iron in the form of transferrin-bound iron
(TBI) and NTBI deposits primarily in the parenchymal cells of the liver. Previous studies
have demonstrated that NTBI uptake is increased in isolated hepatocytes from Hfe
knockout mice (Chua et al. 2004) and decreased in the presence of HFE in Chinese
Hamster Ovary cells (Carlson et al. 2005) and HepG2 hepatoma cells (Gao et al. 2008).
Furthermore, it has recently been demonstrated that there is a link between plasma NTBI
levels and the degree of liver injury in mouse models of HH (Delima et al. 2012). Thus the
relationship between plasma NTBI levels, hepatic NTBI uptake and the pathogenesis of
liver injury in HH is of importance.
In the current study Hfe knockout (Hfe-/-)(Zhou et al. 1998), Tfr2 mutant (Tfr2mut)(Fleming et
al. 2002) and double mutant (Hfe-/-xTfr2mut)(Delima et al. 2012) mouse models of HH were
used to investigate the transport of NTBI in vivo.
4.2 Methods
4.2.1 Animals Animal models were generated and raised as previously described (Materials and
methods 2.2.1)
4.2.2 NTBI uptake NTBI uptake was measured in vivo using a modified method described previously by
Craven et al. (Craven et al. 1987). Based on pre-experiment measurement of plasma total
iron binding capacity and transferrin saturation (Fig 4.1A), plasma transferrin was
saturated with non-radioactive ferric citrate (4-15 nmoles; 1:5 ratio of iron to citrate in 50 µL
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isotonic saline) in Hfe-/-, Tfr2mut and WT mice via lateral tail vein injection. Hfe-/-xTfr2mut
double mutant mice received an equivolume injection of isotonic saline as plasma
transferrin was already saturated with iron. After 15 min, the mice were injected with NTBI
in the form of 59Fe-citrate (0.6 nmole; 1:5 ratio of iron to citrate in 30 µL isotonic saline) via
lateral tail vein and blood samples (50 µL) were collected at various time points. After 30
min, blood was collected by cardiac puncture, and mice were perfused with 15-20 mL ice-
cold 0.15 M saline in situ after which the liver, kidneys, pancreas, heart, duodenum and
femurs were collected and counted for radioactivity using a Wizard gamma counter
(PerkinElmer, Massachusetts USA). Specific activity of 59Fe-NTBI was corrected to
account for the different pool sizes of circulating NTBI in HH and WT mice using the post-
experiment plasma NTBI concentration (Fig. 4.1C). The amount of 59Fe-NTBI in the
plasma samples taken at various time intervals after 59Fe-citrate injection was expressed
as a percentage of the zero time value to determine the plasma NTBI clearance. Tissue
NTBI uptake was calculated using tissue 59Fe content and corrected specific activity and
expressed as pmoles NTBI per total organ or g wet weight.
4.2.3 Tissue collection Tissues used in this study were collected as stated in Materials and methods 2.2.1.
4.2.4 Plasma iron measurement Blood was collected by cardiac puncture according to the method previously described
(Materials and Methods 2.2.1). Plasma iron assays were conducted according to the
methods described in Materials and Methods 2.2.3-5.
4.2.5 Hepatic iron content Hepatic iron content was measured by ICP-AES as previously described (Materials and
methods 2.2.2)
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4.2.6 Statistics Results are expressed as mean ± SEM, n=6 and were calculated as previously described
(Materials and methods 2.2.13).
4.3 Results
4.3.1 Tissue iron content Liver iron concentration was elevated in all HH and dietary iron-loaded mice compared
with non-iron-loaded WT mice. Liver iron levels in Hfe-/- and iron-loaded WT mice were
similar and more than 3-fold higher than non-iron-loaded WT mice (p<0.001; Table 4.1).
Tfr2mut mice exhibited significantly (p<0.01) higher HIC than Hfe-/- mice. Furthermore, iron
concentration in the liver of Hfe-/-xTfr2mut mice was approximately 1.5-fold greater than Hfe-
/- or Tfr2mut mice (Table 4.1), and approximately 5-fold higher than non-iron-loaded WT
mice, consistent with previously reported results using a colorimetric assay (Delima et al.
2012). Iron concentration in the kidneys was similar in Hfe-/-, Tfr2mut and non-iron loaded
WT mice but was less than iron-loaded WT mice (p<0.05; Table 4.1). In Hfe-/-xTfr2mut mice,
kidney iron concentration was increased compared with all other types of mice (p<0.05;
Table 4.1). Pancreatic iron concentration was increased in Hfe-/- and iron-loaded WT mice
(p<0.01), whereas iron concentration in the heart was not increased in these mice
compared with non-iron-loaded WT mice (Table 4.1). In Tfr2mut mice, pancreatic and
cardiac iron levels were more than 4-fold higher than Hfe-/- and WT mice (p<0.01; Table
4.1). Most notably, Hfe-/-xTfr2mut mice had pancreatic and cardiac iron levels approximately
3-fold higher than Tfr2mut mice and more than 10 to 20-fold higher than other types of mice
(p<0.01; Table 4.1).
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Table 4.1: Tissue iron content.
Tissue WT WT+Fe Hfe-/- Tfr2
mut Hfe-/-xTfr2
mut
Liver 2.77 ± 0.04 10.41 ± 0.29a 9.45 ± 0.29
a 11.25 ± 0.57
a,c 14.39 ± 1.00
a,b,c,d
Kidney 1.29 ± 0.05 1.77 ± 0.07a 1.00 ± 0.07
b 1.21 ± 0.02
b 2.79 ± 0.23
a,b,c,d
Pancreas 0.50 ± 0.02 0.80 ± 0.09a 0.88 ± 0.09
a 3.52 ± 0.29
a,b,c 10.02 ± 0.73
a,b,c,d
Heart 0.43 ± 0.04 0.32 ± 0.04 0.43 ± 0.04 1.91 ± 0.09a,b,c
4.70 ± 0.32a,b,c,d
NOTE. Results are expressed as mean ± SEM µmol Fe/g wet weight tissue (n=4-12). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
4.3.2 Plasma iron parameters Plasma TS and NTBI concentrations were increased in all HH and iron-loaded WT mice
compared with non-iron-loaded WT mice (non-hatched bars, p<0.05; Fig. 4.1A, B). The
increases in plasma TS and NTBI levels were greatest in Hfe-/-xTfr2mut mice (non-hatched
bars, p<0.05, Fig. 4.1A, B). NTBI concentration was approximately 4-fold higher than in
either Hfe-/- or Tfr2mut mice (p<0.05, Fig. 4.1B). There was a strong positive correlation
between plasma TS and NTBI concentration (r=0.91, p=0.03; Fig. 4.1C). At the conclusion
of the experiment, TS was approximately 100% in all groups of mice (Fig. 4.1A; hatched
bars). There were also no changes in plasma NTBI levels post-experiment in HH and iron-
loaded WT mice except in non-iron-loaded WT mice (Fig. 4.1B; non-hatched bars versus
hatched bars).
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Figure 4.1: Plasma iron parameters. Transferrin saturation (A) and non-transferrin bound iron (NTBI) concentration (B) were measured pre- (non-hatched bars) and post-NTBI uptake experiment (hatched bars) in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe knockout (Hfe-/-), Tfr2 mutant (Tfr2mut) and Hfe-/-xTfr2mut mice. Correlation between plasma transferrin saturation and plasma NTBI concentration (C). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups. *, p<0.05 versus pre-experiment (non-hatched bars).
r=0.91 p=0.03
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4.3.3 Plasma NTBI clearance NTBI was rapidly cleared from the plasma in Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut, with the
majority of 59Fe-NTBI cleared from the plasma by 2 mins (Fig. 4.2). The clearance on NTBI
was significantly (p<0.001) increased in HH and iron-loaded mice compared with non-iron-
loaded WT mice (Fig. 4.2). Clearance on NTBI from the plasma was most efficient in Hfe-/-
xTfr2mut and Tfr2mut mice was up to 5-fold greater than in non-iron-loaded WT mice at 30
mins. NTBI clearance was approximately 2.5-fold higher in Hfe-/- and iron-loaded WT mice
than in non-iron-loaded WT mice at 30 mins (Fig. 4.2).
Figure 4.2: Plasma NTBI clearance Plasma NTBI clearance was measured at 2 and 30 min in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe knockout (Hfe-/-), Tfr2 mutant (Tfr2mut) and Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=5-8). Clearance at time point: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. *, p<0.05 30 min vs 2 min.
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4.3.4 Tissue NTBI uptake In WT and Hfe-/-xTfr2mut mice, NTBI was taken up predominately by the liver with less
taken up by the kidneys, pancreas, heart and duodenum (p<0.05; Fig. 4.3A), with a similar
pattern of distribution in Hfe-/- and Tfr2mut mice (p<0.05; Fig. 4.3B).
Figure 4.3: Tissue NTBI uptake. Tissue distribution of NTBI uptake was measured in WT mice (dark green bars) and Hfe-/-
xTfr2mut mice (red bars) (A), and Hfe-/- (orange bars) and Tfr2mut mice (light green bars) (B). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus liver; b, p<0.05 versus kidney; c, p<0.05 versus pancreas; d, p<0.05 versus heart denote significance between groups. *, p<0.05 versus WT (green bars) or Hfe-/- (orange bars).
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Liver NTBI uptake was elevated in all iron-loaded mice compared with non-iron-loaded WT
mice (p<0.001). In Hfe-/- mice, liver NTBI uptake was similar to iron-loaded WT mice and
more than 2-fold higher than non-iron-loaded WT mice (p<0.001; Fig. 4.4A). NTBI uptake
was increased by 30% in Tfr2mut mice and by more than 5-fold in Hfe-/-xTfr2mut mice
compared with Hfe-/- mice (p<0.05; Fig. 4.4A). There was a significant positive correlation
between liver NTBI uptake and both liver iron concentration (r=0.75, p<0.0001; Fig. 4.4B)
and plasma NTBI concentration post-experiment (r=0.92, p<0.0001; Fig. 4.4C).
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Figure 4.4: Liver NTBI uptake. Liver NTBI uptake (A) was measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice and plotted against liver iron concentration (B), and plasma NTBI concentration (C). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
r=0.75 p<0.0001
r=0.92 p<0.0001
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NTBI uptake by the kidneys and pancreas was increased in all iron-loaded mice compared
with non-iron-loaded WT mice (Fig. 4.5A and 4.6A; p<0.05). In both kidney and pancreas,
NTBI uptake was similar in Hfe-/- and iron-loaded WT mice, and was increased by 1.5- fold
in Tfr2mut and by 4-5-fold in Hfe-/-xTfr2mut mice compared with Hfe-/- mice (Fig. 4.5A and
4.6A; p<0.05). Cardiac NTBI uptake was similar in Hfe-/- and WT mice, and was
significantly (p<0.01) increased by approximately 70% in Tfr2mut mice, and 4-fold in Hfe-/-
xTfr2mut mice compared with non-iron-loaded mice (Fig. 4.7A). Duodenal NTBI uptake was
similar in Hfe-/-, Tfr2mut and WT mice and increased by more than 3-fold in Hfe-/-xTfr2mut
mice compared with all other groups (Fig. 4.8A; p<0.001). NTBI uptake by the femurs was
considerably lower than NTBI uptake by the liver, kidney, pancreas and heart. Femur NTBI
uptake in Hfe-/- and Tfr2mut mice was reduced by 50%, and increased in Hfe-/-xTfr2mut by 4-
fold compared with non-iron-loaded WT mice (Fig. 4.8B). NTBI uptake was strongly
correlated with plasma NTBI and tissue iron content in the kidneys (r=0.84, p<0.0001
versus iron content, Fig. 4.5B; r=0.86, p<0.0001 versus plasma NTBI, Fig. 4.5C;),
pancreas (r=0.98, p<0.0001 versus iron content, Fig. 4.6B; r=0.89, p<0.0001 versus
plasma NTBI, Fig. 4.6C) and heart (r=0.89, p<0.0001 versus iron content, Fig. 4.7B;
r=0.95, p<0.0001 versus plasma NTBI, Fig. 4.7C) in all groups of mice.
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Figure 4.5: Kidney NTBI uptake. Kidney NTBI uptake (A) was measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice and plotted against liver iron concentration (B), and plasma NTBI concentration (C). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
r=0.84 p<0.0001
r=0.86 p<0.0001
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Figure 4.6: Pancreas NTBI uptake. Pancreas NTBI uptake (A) was measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice and plotted against liver iron concentration (B), and plasma NTBI concentration (C). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
r=0.98 p<0.0001
r=0.89 p<0.0001
100
Figure 4.7: Heart NTBI uptake. Heart NTBI uptake (A) was measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-
xTfr2mut mice and plotted against liver iron concentration (B), and plasma NTBI concentration (C). Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
r=0.89 p<0.0001
r=0.95 p<0.0001
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Figure 4.8: Duodenum and femur NTBI uptake Duodenum NTBI uptake (A) and Femur NTBI uptake (B) were measured in WT, WT+Fe, Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=5-11). a, p<0.05 versus WT; b, p<0.05 versus WT+Fe; c, p<0.05 versus Hfe-/-; d, p<0.05 versus Tfr2mut denote significance between groups.
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4.4 Discussion In this study, NTBI transport and tissue uptake in mouse models of HH were examined.
NTBI was cleared rapidly from the circulation in all mouse models of HH, with most of the
NTBI taken up by the liver and to a lesser degree by the kidneys, pancreas and heart. The
current study describes for the first time increased in vivo tissue NTBI uptake in mouse
models of HH with the greatest increase observed in Hfe-/-xTfr2mut mice followed by Tfr2mut
and Hfe-/- mice (Hfe-/-xTfr2mut >> Tfr2mut > Hfe-/- mice) compared with non-iron-loaded wild-
type mice. There was a significant positive correlation between NTBI uptake and both
plasma NTBI levels and iron content in the liver, kidney, pancreas and heart. This
suggests that NTBI uptake is likely to contribute to excessive iron deposition primarily in
the liver as well as in the kidney, pancreas and heart in HH.
In vivo transport of NTBI was determined in murine HH models in the presence of
circulating transferrin. The observation that the iron-binding capacity of transferrin was
saturated post-experiment demonstrated that the injection of known quantities of non-
radioactive ferric citrate saturated circulating plasma transferrin. The absence of significant
changes in plasma NTBI levels pre- and post-experiment in HH mice suggested that the
injection of non-radioactive ferric citrate prior to the experiment was sufficient to saturate
the transferrin without donating additional NTBI to the circulation. As iron citrate is the
predominant form of circulating NTBI in HH (Grootveld et al. 1989), citrate was chosen as
the ligand to examine in vivo NTBI transport in this study. The clearance of plasma 59Fe-
NTBI was rapid in HH mice and occurred significantly faster than in WT mice. This is much
shorter than the plasma half-life for TBI of approximately 50-60 mins that has been
reported previously in both HH (Hfe-/- and Tfr2mut) and WT mice (Chua et al. ; Frazer 2002).
The rapid clearance of plasma NTBI compared with TBI is likely to reflect the relatively
lower concentration of plasma NTBI and the higher capacity of tissue NTBI transporters
compared with transferrin receptors that mediate TBI uptake. Most of the 59Fe-NTBI was
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primarily taken up by the liver, followed by the kidneys, pancreas and heart consistent with
the tissue distribution of NTBI uptake observed previously in vivo in hypotransferrinemic
mice (Craven et al. 1987) and Dmt1flox mice (Wang and Knutson 2013). A greater
proportion of 59Fe was deposited in the livers of HH mice compared with non-iron-loaded
wild-type mice. 59Fe uptake by the femur was very low suggesting that 59Fe was present in
the circulation in the form of NTBI and not TBI, which is cleared predominately by the bone
marrow for erythropoiesis.
HH mice exhibited increased plasma NTBI levels and NTBI clearance, liver NTBI uptake
and liver iron content. These features were most marked in Hfe-/-xTfr2mut mice compared
with either Hfe-/- or Tfr2mut mice. Liver NTBI uptake was strongly associated with both liver
iron content and plasma NTBI levels. However, the marked elevation of these parameters
in the Hfe-/-xTfr2mut mice may influence the observed statistical correlation between plasma
NTBI levels, tissue iron content and NTBI uptake. It have previously demonstrated that
liver TBI uptake in vivo was also increased in the presence of elevated plasma TBI levels
in both Hfe-/- (Frazer 2002) and Tfr2mut mice (Chua et al. 2010). Liver expression of
transferrin receptor 1 (TFR1) is downregulated in HH in response to iron-loading (Chua et
al. 2010) and although TFR2 expression is upregulated in HH in the presence of increased
levels of diferric transferrin (Robb and Wessling-Resnick 2004), previous studies have
shown that TFR2 accounts for only approximately 20% of TBI uptake in vivo (Chua et al.
2010). Hence TBI uptake is likely to play a lesser role than NTBI uptake in liver iron-
loading in HH.
The efficient clearance of NTBI by the liver is well documented, with studies in mice
indicating that 58 to 75% of NTBI is cleared by the liver on its first pass from the portal
circulation and stored in the liver as ferritin (Brissot et al. 1985). Studies by Craven et al. in
transferrin-iron saturated rats (Craven et al. 1987) are consistent with the findings of the
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current study where NTBI delivered intravenously was cleared rapidly from the circulation
and the majority was found deposited in the liver, demonstrating efficient clearance of
plasma NTBI by the liver. The ability for hepatocytes to take up NTBI has also been
demonstrated in vitro in isolated hepatocytes from rats (Baker et al. 1998) and mice, with
an upregulation of NTBI uptake by hepatocytes in Hfe-/- mice (Chua et al. 2004). These
studies demonstrated that hepatocytes take up NTBI via a low-affinity membrane
transporter that is saturable at elevated non-physiological iron concentrations, such as
those found in HH (Barisani et al. 1995). The current study, suggests that liver NTBI
transport in vivo may be positively regulated by iron in HH, consistent with in vitro studies
in fibroblast (Craven et al. 1987) and hepatoma (Randell et al. 1994) cell lines that also
demonstrated increased NTBI uptake with iron-loading. As in HH, NTBI also plays a role in
beta-thalassemia, alcoholism, haematological malignancies, diabetes mellitus (De Feo et
al. 2001; Pootrakul et al. 2004; Lee et al. 2006; Sahlstedt et al. 2009) and end stage renal
disease (Prakash et al. 2005) where excess NTBI is deposited in the liver. Excessive iron
deposition leads to the production of ROS which are linked to liver injury through the
promotion of lipid peroxidation and oxidative damage to DNA and proteins and the
production of factors that promote inflammation and fibrosis in human HH (Olynyk et al.
2008; Ramm and Ruddell 2010).
It is well established that divalent metal transporter 1 (DMT1) is integral in the uptake of
dietary NTBI in the duodenum (Gunshin et al. 1997). In the liver, several NTBI transporters
have been identified, with uptake of NTBI in hepatocytes and hepatoma cells mediated by
DMT1(Shindo et al. 2006) and Zrt-Irt-like protein 14 (ZIP14) (Liuzzi et al. 2006). The
presence of HFE has been shown to decrease ZIP14 levels by reducing protein stability
resulting in decreased NTBI uptake (Gao et al. 2008). The current observation in HFE
deficient mice (Hfe-/- and Hfe-/-xTfr2mut) of increased liver NTBI uptake in vivo are
consistent with the previous in vitro data and a negative regulatory role for HFE in NTBI
105
uptake. Dmt1 protein expression has been reported to be increased (Trinder et al. 2002) or
decreased (Nam et al. 2013), whilst Zip14 protein expression was increased (Nam et al.
2013) in the liver of mice with iron loading. Specific deletion of Dmt1 in the liver however,
did not alter liver NTBI uptake or liver iron levels suggesting that DMT1 is not essential for
hepatic NTBI uptake (Wang and Knutson 2013). Global deletion of Zip14 in mice has been
reported to have unchanged (Hojyo et al. 2011) or increased liver iron levels (Beker
Aydemir et al. 2012) however liver NTBI uptake was not measured in these studies.
Typically the kidney is unaffected in HH (Pietrangelo 2004), however there are reports of
kidney iron overload in cases of severe and lethal idiopathic neonatal haemochromatosis
(Herrmann et al. 2004), The Hfe-/-xTfr2mut mouse model represents a severe form of HH
(Chua et al. 2010) which exhibits higher NTBI uptake and greater deposition of iron in the
kidney than WT mice. Under normal physiological conditions, the kidney acquires iron by
receptor-mediated endocytosis of TBI (Zhang et al. 2007). However, in HH as transferrin
becomes saturated, free iron will bind to low-molecular weight ligands which may be
filtered by the glomerulus in the kidney. The iron transporter DMT1 is highly expressed in
the renal medulla and at the brush border and apical pole endothelial cells in the proximal
tubule (Abboud and Haile 2000). However, it has been shown that the kidney is comprised
predominantly of DMT1 that contains an iron regulatory element (Canonne-Hergaux and
Gros 2002), which is likely to result in the downregulation of DMT1 protein in the presence
of high renal iron levels. Therefore, it is unlikely that DMT1 plays a major role in renal iron-
loading in HH and is more likely a mechanism for iron scavenging from the urine in
conditions of iron deficiency. Lipocalin 2 or m24p3 is a component of the innate immune
system that binds and sequesters bacterial iron compounds in the blood and urine and is
heavily expressed in the kidney and epithelial tissues (Schmidt-Ott et al. 2006). Though its
role in times of infection is well characterised (Yang et al. 2002), it may also play a role in
the transport of NTBI during normal iron homeostasis and possibly in HH (Nairz et al.
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2009).
As in chronically transfused thalassemia major patients (Noetzli et al. 2009), HH patients
with severe iron overload may develop iron-related dysfunction of the heart and pancreas
(Camaschella and Poggiali 2009). Similar results are found in the hypotransferrinemic
mouse where NTBI is the predominant form of circulating iron (Craven et al. 1987). NTBI
uptake by the pancreas and heart is thought to share a similar mechanism. ZIP14 is
expressed in the heart and pancreas (Taylor et al. 2005) and may play a role in pancreatic
and cardiac NTBI uptake. Furthermore, L-type voltage-dependent calcium channels are
highly expressed in the heart and have been shown to facilitate NTBI uptake by
cardiomyocytes (Oudit et al. 2003). L-type calcium channels are also highly expressed in
the pancreas and may contribute to NTBI uptake by pancreatic cells (Lipscombe et al.
2004).
In conclusion, this study clearly demonstrates that plasma NTBI concentration and
clearance as well as in vivo tissue NTBI uptake were significantly increased in all three
mouse models of HH. Changes in these NTBI parameters were greatest in Hfe-/-xTfr2mut
mice followed by Tfr2mut and Hfe-/- mice (Hfe-/-xTfr2mut >> Tfr2mut > Hfe-/- mice) compared
with non-iron-loaded wild-type mice. The positive linear relationship between NTBI uptake
and both plasma NTBI and tissue iron concentrations in the liver, kidney, pancreas and
heart suggests that elevated NTBI uptake from the plasma contributes to the excessive
iron deposition in the liver and to a lesser degree in the kidney, pancreas and heart in HH.
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Chapter 5
Disruption of HFE and TFR2 causes iron-induced liver
injury in mice
108
The following chapter “Disruption of HFE and TFR2 causes iron-induced liver injury in
mice” has been published as:
Delima, R. D., A. C. Chua, et al. (2012). "Disruption of HFE and TFR2 causes iron-induced
liver injury in mice." Hepatology. 56(2):585-93.
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5.1 Introduction
In hereditary haemochromatosis dysrgulated iron homeostasis results in increased
intestinal iron absorption, leading to increased cellular uptake of the excess circulating iron
which is stored as ferritin and haemosiderin, primarily in the liver, but with increased
severity of iron loading it may also involve other organs. Excessive iron deposition has
been linked to tissue damage and cellular dysfunction. Progressive iron deposition in the
liver leads to fibrosis, cirrhosis and hepatocellular carcinoma with the duration of iron
loading increasing the risk of developing significant liver injury (Olynyk et al. 2005).
Studies have shown that when hepatic iron concentration exceeds 60 µmol/g, hepatic
stellate cells begin to exhibit early signs of activation, an integral event in the initiation of
hepatic fibrosis (Ramm et al. 1997). As hepatic iron levels increase further, the risk of
significant liver fibrosis and ultimately cirrhosis increases (Adams 2001). Although the
exact mechanisms of liver injury induced by iron overload have not yet been fully
elucidated, it is thought that the accumulation of excess iron-catalyzed reactive oxygen
species (ROS) plays a significant role. Previous studies have demonstrated decreased
hepatic levels of antioxidants such as superoxide dismutase (SOD), ascorbate, β-carotene
and vitamins E and A in iron overload conditions (Livrea et al. 1996; Brown et al. 1998).
Furthermore, iron increases the level of lipid peroxidation products, such as
malondialdehyde and F2-isoprostanes (Matayatsuk et al. 2007), which can cause
mutagenesis in DNA (el Ghissassi et al. 1995). Lipid peroxidation-induced DNA lesions are
increased two- to three-fold in the livers of HH patients and, together with the iron overload
seen in HH are associated with an approximately 20-fold increased risk of hepatocellular
carcinoma (Elmberg et al. 2003). Oxidative stress has been shown to activate apoptosis
and necrosis, promoting the synthesis and release of pro-inflammatory and fibrogenic
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factors that alter Kupffer cell and hepatocyte functions, triggering the activation of hepatic
stellate cells and fibrogenesis (Olynyk et al. 2008).
There is a clear association between increased hepatic iron concentration (HIC) and
fibrosis stage (Olynyk et al. 2005), and whilst several hypotheses exist on the mechanisms
responsible for hepatocellular injury, the precise pathways associated with liver cell
dysfunction and fibrogenesis remain to be fully elucidated, in part due to an absence of
suitable models.
5.2 Methods
5.2.1 Animals Animal models were generated and raised as previously described (Materials and
methods 2.2.1)
5.2.2 Tissue collection Liver tissue was collected as described in Materials and methods 2.2.1
5.2.3 Histology Liver tissue for immunohistochemistry was fixed in 10% neutral buffered formalin overnight
before being subjected to routine histological processing. Four µm liver sections were cut
by microtome and mounted on SuperFrost Plus (Menzel-Gläser, Germany) glass slides.
Liver tissue for immunofluorescence was immediately embedded in Tissue-Tek® OCT™
Compound (Sakura, The Netherlands) and frozen in liquid nitrogen. Seven µm liver
sections were cut by cryostat and mounted on SuperFrost Plus (Menzel-Gläser, Germany)
glass slides and stored at -80°C.
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5.2.4 Perls' Prussian blue staining Liver sections were dewaxed in two changes of xylene for 5 min, and then two changes of
100% ethanol for 2 min, and two changes of 75% ethanol for 5 min before being placed in
running water. Slides were then rinsed in distilled water and then placed in the prussian
blue reaction solution (2% potassium ferrocyanide : 2% hydrochloric acid) for 10-20 min.
After incubation in Prussian blue solution, the slides were rinsed in distilled water and
counterstained in 1% neutral red for 30 seconds. Once counterstained, the slides were
rinsed in distilled water and then dehydrated in two changes of 75% ethanol for 2 min
each, two changes of 100% ethanol for 2 min each, followed two changes of xylene for 5
min each. The slides were then mounted using the DePeX Mounting Medium and
coverslips.
5.2.5 Haemotoylin & Eosin staining Staining was performed on formalin-fixed paraffin embedded (FFPE) sections according to
standard histopathological methods.
5.2.6 Immunofluorescence Cluster of differentiation 45–positive (CD45+) staining was performed on methanol/acetone
(1:1) fixed liver cryosections using a rat anti-CD45 antibody (Ly-5, 1:150; BD Pharmingen,
SanDiego, CA) and detected with goat anti-rat Alexa Fluor 594 or goat antirat Alexa Fluor
488 (1:200; Invitrogen, Mulgrave, Victoria, Australia) and mounted with Long Gold antifade
reagent, containing 40,6-diamidino-2-phenylindole (DAPI; Invitrogen), for nuclear
quantitation. Quantification was performed by the acquisition of six random, non-
overlapping fields of view per tissue sample, followed by colocalization analysis of CD45
and DAPI (nuclear quantification) using the AnalySIS Life Science Professional program
(Olympus, Melbourne, Victoria, Australia). Ferritin staining was performed using a rabbit
anti-ferritin antibody (1:800; Dako, Glostrup, Denmark) and detected using a goat anti-
rabbit Alexa Fluor 594 (1:200; Invitrogen).
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5.2.7 Biochemical markers of liver injury Plasma alanine aminotransferase (ALT) was measured as an indicator of liver injury using
a kit according to the manufacturer’s instructions (Sigma Chemical Company, MO). Liver
F2-isoprostanes, a marker of lipid peroxidation, was measured using gas chromatography-
mass spectrometry using a deuterium-labeled internal standard as previously described
(Mori et al. 1999) by Professor Kevin Croft (School of Medicine and Pharmacology, Royal
Perth Hospital, UWA). The antioxidant butylated hydroxyl toluene was added to the liver
tissue to scavenge any ROS generated during tissue storage and processing. Lipid
peroxidation was also examined by measuring liver thiobarbituric acid reactive species
(TBARS), which consists predominately of the lipid peroxidation product, malondialdehyde
(MDA), using a kit according to the manufactures’ instructions (Cayman Chemical,
Sydney, Australia). As an indicator of oxidative stress the activities of the antioxidant
enzymes copper/zinc and manganese superoxide dismutase (SOD) were measured in
liver homogenate using a kit according to the manufacturer’s instructions (Cayman
Chemical, Sydney, Australia). Liver hydroxyproline content was measured colourimetrically
as a biochemical marker of liver collagen using acid hydrolyzed liver samples, according to
the manufacturer’s instructions (QuickZyme Biosciences, Leiden, Netherlands).
5.2.8 Collagen staining Staining for collagen deposition was performed on FFPE liver sections. Slides were heated
at 60°C for 1hr. Sections were then deparaffinised in Xylene and brought to water via a
decreasing ethanol series. For Sirius Red staining, sections are initially immersed in a
0.1% solution of Sirius Red (Sigma-aldrich, Australia) in saturated aqueous picric acid
(Sigma-aldrich, Australia) for 1 h. Sections were rinsed briefly in distilled water before
immersion in a 0.1% solution of Fast Green (Sigma-aldrich, Australia). Slides were rinsed
in distilled water, dehydrated in ethanol and cleared in xylene, before being mounted with
DePex (VWR, Australia).
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For Masson’s Trichrome stain for collagen deposition, after being brought to water,
sections were stained in Weigert’s Iron Haematoxylin for 10 mins. Sections were washed
in warm tap water and then distilled water, followed by staining in Biebrich Scarlet-Acid
Fuchsin, washing in distilled water and differentiation in Phosphomolybdic-
Phosphotungstic acid solution (5% Phosphomolybdic acid : 5% Phosphotungstic acid) for
15 mins. Sections were immediately transferred to Aniline Blue solution for 5 mins,
differentiated in 1% acetic acid for 2 mins and washed in distilled water. Sections were
quickly dehydrated in ethanol to avoid removal of Biebrich Scarlet-Acid Fuchsin stain,
cleared in xylene and mounted in DePex (VWR, Australia). Stained sections were digitised
using an Aperio Scanscope XT using a positive pixel count algorithm supplied by the
manufacturer (Aperio Technologies, Vista, CA). Pixel positivity was determined by the
number of pixels representing stained tissue divided by the total number of pixels in the
whole liver section.
5.2.9 Gene expression Measurement of liver gene expression was performed by RT-PCR as described in
Materials and methods 2.2.6-11.
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5.3 Results
5.3.1 Iron measurements Plasma iron levels were measured in Hfe-/-xTfr2mut, Hfe-/-, Tfr2mut, and wild-type mice and
described previously in Chapter 3. Briefly, plasma iron concentration and transferrin
saturation were higher in Hfe-/-xTfr2mut, Tfr2mut, Hfe-/- and iron-loaded WT mice compared
with non-iron-loaded WT mice (Chapter 3; Fig. 1A,B). Plasma iron levels were highest in
Hfe-/-xTfr2mut mice (Chapter 3, Fig. 1A,B) and were significantly greater than Tfr2mut and
Hfe-/- mice. Perls’ Prussian blue staining of liver sections from Hfe-/-xTfr2mut mice
demonstrated a periportal distribution of iron, similar to that seen in Hfe-/-, Tfr2mut and iron-
loaded wild-type mice. However, the intensity of iron staining was greater in Hfe-/-xTfr2mut
than in the other types of mice (Fig. 5.1B-D) as measured by Pixel positivity. These results
indicate an increased iron burden in Hfe-/-xTfr2mut mice and are corroborated by non-haem
iron measurements (Fig. 5.1A).
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Figure 5.1: Hepatic iron concentration. Hepatic iron concentration was determined biochemically and by Perls’ Prussian blue staining. (A) Results are expressed as mean ± SEM (n=5-15). a, p<0.001 vs. WT; b, p<0.001 vs. WT+Fe; c, p<0.01 vs. Hfe-/-; d, p<0.001 vs. Tfr2mut. Staining was conducted on wild-type (WT; B), iron-loaded wild-type (WT+Fe; C), Hfe-/- (D), Tfr2mut (E), and Hfe-/-
xTfr2mut (F) mice. Each panel is representative of staining from 6-8 animals.
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5.3.2 Liver histology H&E-stained liver sections from Hfe-/-xTfr2mut mice demonstrated mild inflammation with
evidence of scattered foci of infiltrating inflammatory cells throughout the liver parenchyma
(Fig. 5.2, indicated by arrows). Immunofluorescent detection of the pan leukocyte marker,
CD45, revealed that the cell aggregates consisted mainly of CD45+ inflammatory cells (Fig.
5.2A, E) that colocalized predominately, but not exclusively, with the iron storage protein,
ferritin, in periportal regions of the liver (Fig. 5.3). The number of CD45+ inflammatory cells
was significantly increased in the livers from Hfe-/-xTfr2mut mice, compared with the other
groups of mice (P<0.05), whereas the number of CD45+ cells in Hfe-/-, Tfr2mut, and iron-
loaded WT mice was not significantly different from those in non-iron-loaded WT mice (Fig.
5.2F). Another unique feature of Hfe-/-xTfr2mut mice was the evidence of inflammatory
sideronecrosis of hepatocytes, which was not observed in any other group of mice (Fig.
5.2E).
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Figure 5.2: Liver histology. H&E staining of liver sections from WT (left panel, A), iron-loaded WT (WT+Fe; B), Hfe-/-
(C), Tfr2mut (D), and Hfe-/-xTfr2mut (left panel, E) mice. Arrows indicate inflammatory sideronecrosis of hepatocytes (left panel, E). CD45-stained (red) liver sections from WT (right panel, A) and Hfe-/-xTfr2mut (right panel, E) mice. Each panel shows a representative photomicrograph of staining from 6 animals. The number of CD45+ cells (F) is expressed as mean ± SEM (n=6). a, P<0.05 versus WT; b, P<0.05 versus WT+Fe; c, P<0.05 versus Hfe-/-; d, P<0.05 versus Tfr2mut.
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Figure 5.3: CD45+ / ferritin double staining in Hfe-/-xTfr2mut mice. Regions of high hepatic iron concentration were identified in the periportal regions of the liver by immunofluorescent staining for the iron storage protein ferritin (Fn; A, D). Clusters of CD45+ inflammatory cells (B, E) were predominantly but not exclusively seen in close spatial contact with Fn+ cells (C, F; merged with DAPI for nuclear staining).
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5.3.3 Biochemical markers of liver injury Liver injury was assessed by examining plasma ALT and hepatic SOD levels. Also lipid
peroxidation was assessed by measuring liver F2-isoprostane and TBARS levels. Plasma
ALT activity was increased in Hfe-/-xTfr2mut mice by at least 1.8-fold, compared with all
other types of mice (P<0.001; Fig. 5.4A). Both hepatic copper/zinc (cytosolic) and
manganese (mitochondrial) SOD activities were decreased significantly in all HH mice. In
Hfe-/-xTfr2mut mice copper/zinc SOD levels were similar, whereas manganese SOD levels
were significantly (p<0.01) lower than Hfe-/-and Tfr2mut mice (Fig. 5.4B). Liver F2-
isoprostanes were elevated in all groups of HH mice, compared with non-iron-loaded WT
mice (p<0.01), with Hfe-/-xTfr2mut mice having similar liver F2-isoprostane levels to iron-
loaded WT mice and significantly (p<0.01) higher levels than either Hfe-/- or Tfr2mut mice
(Fig. 5.4C). Hfe-/-xTfr2mut mice had TBARS levels 30% higher than Hfe-/- and Tfr2mut mice,
which were in turn 25% higher than the wild-type mice (Fig. 5.4D).
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Figure 5.4: Biochemical markers of liver injury. Plasma ALT (A), liver copper/zinc (non-hatched bars) and manganese (hatched bars) SOD (B), liver F2-isoprotane (C), liver TBARS (D), and hydroxyproline (E) levels were measured in WT, WT+Fe, Hfe-/-, Tfr2mut, and Hfe-/-
xTfr2mut mice. Results are expressed as mean ± SEM (n=5-15). a, P<0.05 versus WT; b, P<0.05 versus WT+Fe; c, P<0.05 versus Hfe-/-; d, P<0.05 versus Tfr2mut. For manganese SOD: 1, P<0.05 versus WT; 2, P<0.01 versus WT+Fe; 3, P<0.01 versus Hfe-/-; 4, P<0.01 versus Tfr2mut.
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5.3.4 Collagen deposition Hepatic collagen deposition, a marker of fibrosis, was examined by histology using Sirius
red and Masson’s Trichrome staining and by biochemical measurement of hydroxyproline
levels. Hydroxyproline levels were increased in all iron-loaded mice. In Hfe-/-xTfr2mut mice,
hydroxyproline levels were significantly increased compared with Tfr2mut mice and both
were elevated compared with Hfe-/- and iron-loaded wild-type mice (Fig. 5.5E; p<0.05).
Likewise, Hfe-/-xTfr2mut mice had significantly (p<0.05) increased Sirius red staining
compared with Hfe-/-, Tfr2mut and iron-loaded wild-type mice, which in turn exhibited greater
collagen deposition than non-iron-loaded wild-type mice (p<0.01; Fig. 5.5A-F). Sirius red
staining revealed portal tract thickening and periportal fibrosis, and there was evidence of
portal tract bridging in Hfe-/-xTfr2mut mice, which was not evident in other groups.
Quantification of Sirius red staining correlated with HIC (r2=0.98, p=0.001; Fig. 5.6A),
plasma NTBI (r2=0.82, p=0.033; Fig. 5.6B) as well as hydroxyproline (r2=0.89, p=0.015;
Fig. 5.6C) and F2-isoprotane levels (r2=0.77, p=0.048; Fig. 5.6D) in HH mice. This
suggests that biochemical measurements of collagen levels measured were consistent
with histological observations using Sirius red staining and were dependent on both
plasma NTBI and HIC in HH mice. Furthermore, the intensity of Trichrome staining, a
commonly used but less sensitive marker of fibrosis, was also significantly enhanced in
Hfe-/-xTfr2mut and Tfr2mut mice (Fig. 5.7F) with evidence of collagen thickening in the
periportal region of the liver (Fig. 5.7A-E).
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Figure 5.5: Liver collagen deposition via Sirius red stain. Sirius red staining of liver sections from WT (A), iron-loaded WT (WT+Fe; B), Hfe-/-(C), Tfr2mut (D), and Hfe-/-xTfr2mut (E) mice. Staining intensity is quantified in (F). There was increased collagen deposition in the portal tracts of WT+Fe, Hfe-/-, and Tfr2mut mice with advanced thickening of the portal tract in Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=6). a, P < 0.01 versus WT; b, P<0.05 versus WT+Fe; c, P<0.01 versus Hfe-/-; d, P<0.05 versus Tfr2mut. Each panel is a representative photomicrograph of staining from 6 animals.
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Figure 5.6: Sirius red stain correlated with iron, collagen and lipid peroxidation measurement. Sirius red staining of liver sections from WT, iron-loaded WT, Hfe-/-, Tfr2mut, and Hfe-/-
xTfr2mut mice was correlated with hepatic iron concentration (A), plasma non-tranferrin bound iron concentration (B), liver hydroxyproline content (C), and F2-Isoprostane levels (D). Each data point is expressed as mean ± SEM (n=6).
r2=0.98
p=0.001 r2=0.82
p=0.033
r2=0.77
p=0.048 r2=0.89
p=0.015
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Figure 5.7: Liver collagen deposition using Masson’s trichrome stain. Trichrome staining of liver sections from WT (A), WT+Fe B), Hfe-/-(C), Tfr2mut (D), and Hfe-/-
xTfr2mut (E) mice. Staining intensity is quantified in (F). There was increased collagen deposition in the portal tracts of WT+Fe, Hfe-/-, and Tfr2mut mice with advanced thickening of the portal tract in Hfe-/-xTfr2mut mice. Results are expressed as mean ± SEM (n=6). a, P < 0.01 versus WT; b, P<0.05 versus WT+Fe; c, P<0.01 versus Hfe-/-; d, P<0.05 versus Tfr2mut. Each panel is a representative photomicrograph of staining from 6 animals.
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5.3.5 Hepatic expression of injury-related genes The hepatic expression of the injury-related genes c-Myc, IL-10, and Tnfα were measured
via RT-PCR as were the cytokines (described in detail in Chapter 6; Fig. 6.2A, B, C) IL-1α,
IL-6 and IL-17. c-Myc expression was significantly (p<0.05) higher in Hfe-/-xTfr2mut mice
compared with the other HH and wild-type mice. c-Myc expression in Hfe-/-xTfr2mut mice
was 2-fold higher than Tfr2 mice, which in turn, was 2-fold higher than Hfe and non-iron
loaded wild-type mice and 3-fold higher than non-iron-loaded wild-type mice (Fig. 5.8A).
IL-10 mRNA in Hfe-/-xTfr2mut andTfr2mut mice was very low compared with wild-type mice.
Hfe-/- mice had IL-10 levels similar to that of wild-type mice, whilst expression in iron-
loaded wild-type mice was significantly (p<0.05) higher than all other groups of mice (Fig.
5.8B). As described in detail in Chapter 6; Fig. 6.2D liver expression of Tnfα was similar in
Hfe-/, Tfr2mut and wild-type mice and was more than 2-fold lower than in Hfe-/-xTfr2mut mice
(p<0.05; Fig. 5.8C).
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Figure 5.8: Liver expression of injury-related genes.
mRNA expression was determined by real-time qPCR for c-Myc (A), IL-10 (B) and Tnfα (C) in wild-type (WT), WT+Fe, Hfe-/-, (Tfr2mut) and Hfe-
/-xTfr2mut mice. Results are expressed as mean ± SEM (n=4-8). Saline: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut.
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5.4 Discussion In this study, Hfe-/- and Tfr2mut mouse models of HH types 1 and 3, respectively, and a Hfe-
/-xTfr2mut mouse model were used to examine the effects of disruption of Hfe and Tfr2,
either alone or in combination, on iron-induced liver injury. The Hfe-/-xTfr2mut mouse is the
first report of a genetic HH mouse model of iron-induced liver injury, which reflects both the
iron-loaded phenotype and increased liver injury seen in HH patients.
As previously discussed Hfe-/-xTfr2mut mice had elevated plasma and hepatic iron levels,
determined by both biochemical (Chapter 3; Fig. 3.1-2) and histological methods (Chapter
5; Fig. 5.1), compared with Hfe-/- and Tfr2mut mice. In association with increased liver iron
loading, there was a pronounced elevation of plasma ALT activity, a marker of liver injury,
in Hfe-/-xTfr2mut mice. There was also mild hepatic inflammatory cell infiltration with
scattered foci of CD45+ leukocytes and some evidence of hepatocyte sideronecrosis in
Hfe-/-xTfr2mut mice. Elevated hydroxyproline levels, and Sirius red and Trichrome staining
demonstrating marked portal tract collagen deposition and portal bridging in Hfe-/-xTfr2mut
mice strongly supports the presence of liver fibrosis and was consistent with areas of high
iron accumulation. In comparison, Hfe-/- and Tfr2mut mice had less collagen deposition and
inflammation. Histological evidence of a more pronounced liver damage in Hfe-/-xTfr2mut
mice was corroborated by decreased SOD activity and enhanced lipid peroxidation in the
liver, indicating elevated hepatic oxidative stress.
Mice with deletions in both Hfe and Tfr2 have been generated on other genetic
backgrounds (Corradini et al. 2011; Wallace et al. 2011). Other models of HFE/TFR2
disruption, similar to the Hfe-/-xTfr2mut murine model, exhibited elevated plasma and liver
iron levels compared with mice with the appropriate deletion of Hfe or Tfr2. The degree of
iron overload however, varies between strains, which is consistent with previous
observations that iron metabolism is modified by genetic background (McLachlan et al.
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2011). The Hfe-/-xTfr2mut HH mouse was generated on an AKR background, which has
relatively high iron levels (McLachlan et al. 2011) and this is likely to contribute to the more
advanced pathology of iron overload-induced liver injury seen in the Hfe-/-xTfr2mut mice
compared with HFE/TFR2 mouse models on other background strains.
Rodents are generally relatively resistant to iron-induced liver injury. Dietary carbonyl iron
loading of rats for 3 months produced iron loading in hepatocytes, similar to the levels
seen in the Hfe-/-xTfr2mut mice in the present study, but demonstrated only early signs of
liver injury including increased lipid peroxidation and collagen gene expression. Long-term
iron loading was required for up to 12 months before morphological evidence of fibrosis
was observed (Pietrangelo et al. 1990; Britton et al. 1994). Dietary iron supplementation in
combination with hepatotoxins such as ethanol and carbon tetrachloride was required to
accelerate liver injury (Mackinnon et al. 1995; Lakshmi Devi and Anuradha 2010). In the
present study, the degree of liver fibrosis seen in Hfe-/-xTfr2mut mice at 3 months of age
was similar to that observed after dietary iron-loading of rats for 12 months (Pietrangelo et
al. 1990; Britton et al. 1994). In the Hfe-/-xTfr2mut mice hepatic inflammation, fibrosis and
lipid peroxidation occurred in the presence of marked elevation of both plasma NTBI and
HIC similar to those observed in human HFE-HH (Bacon et al. 1999; Breuer et al. 2000).
Furthermore, the degree of fibrosis seen in the HH mice was dependent on both HIC and
NTBI levels.
The observation that Hfe-/-xTfr2mut mice have increased plasma ALT levels is consistent
with previous observations in HH patients, where the majority of patients had mildly
elevated ALT levels (Lin and Adams 1991). Levels of the anti-oxidant enzymes, cytosolic
copper/zinc and mitochondrial manganese SOD, were both decreased in Hfe-/-xTfr2mut
mice consistent with increased oxidative stress. Earlier studies have also reported
decreased copper/zinc SOD in dietary iron-loaded animals while manganese SOD was
129
decreased in Hfe knockout and increased in iron-loaded rodents (Brown et al. 1998;
Jouihan et al. 2008; Tan et al. 2011). Furthermore, lipid peroxidation was increased in HH
mice. Unexpectedly, the level of F2-isoprostanes in dietary iron-loaded mice was greater
than in HH mice with similar HIC. This may be due to differences between dietary iron
(high hepcidin) and genetic HH (low hepcidin) models of liver iron overload where variation
in cellular iron distribution between parenchymal and Kupffer cells occurs despite similar
total HIC. Though both F2-isoprostanes and TBARS show elevated levels of lipid
peroxidation in Hfe-/-xTfr2mut, Hfe-/- and Tfr2mut mice, differences in their findings may be
due the nature of the assay, where TBARS is a measure of varying lipid peroxidation
products, F2-isoprostane meaurements assay a specific lipid peroxidation product.
Mild liver inflammation was observed only in Hfe-/-xTfr2mut mice, suggesting there was an
iron concentration threshold effect. Mild inflammation has been documented in human HH
studies during the development of fibrosis and cirrhosis (Brunt 2005). Deugnier and
colleagues reported inflammatory infiltrates in approximately 50% of liver biopsies from HH
patients (Deugnier et al. 1992). Inflammation was predominantly present in portal and
periportal regions and correlated with histological iron scores, sideronecrotic changes in
hepatocytes and hepatic fibrosis. Another study showed that approximately 25% of liver
biopsies from untreated HH patients displayed moderate inflammatory infiltration (Stal et
al. 1995). Bridle et al. also reported that 60% of liver biopsies from HH patients showed
mild inflammation consisting of scattered inflammatory foci. Furthermore, patients with
hepatic inflammation had a higher incidence of hepatic fibrosis (Bridle et al. 2003). Iron-
loaded and apoptotic/necrotic hepatocytes are purported to induce the activation of hepatic
stellate cells via various signaling mechanisms resulting in enhanced production of
proinflammatory (IL-6, IL-1β, and TNFα) and profibrogenic cytokines (such as TGF-β1) as
well as the recruitment of inflammatory cells (Ramm and Ruddell 2010). This inflammatory
cytokine response has also been shown to stimulate the transcription of c-Myc, through
130
the phosphorylation of Smad3 (Matsuzaki 2012), resulting in increased proliferation and
regeneration of hepatocytes, in response to liver injury. The absence of antifibrogenic IL-
10 in Hfe-/-xTfr2mut mice is likely to exacerbate liver injury in these mice. This study
provides further support for direct hepatotoxic effects of iron overload, which results from
the disruption of Hfe and Tfr2, manifesting as increased inflammation and increased
collagen deposition.
Iron plays an important part in the progression of hepatic injury, and it does this via its
ability to catalyze the formation of highly reactive and damaging ROS. ROS induces tissue
injury by promoting lipid peroxidation as well as protein and DNA modification leading
ultimately to apoptosis and necrosis. Further investigation into the molecular mechanisms
of iron toxicity and how it causes liver injury will provide a better understanding of the role
iron plays in the progression of liver disease. The Hfe-/-xTfr2mut mouse represents a model
of HH that mimics both iron overload and consequent liver injury observed in humans with
HH.
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Chapter 6
Inflammation in mouse models of hereditary
haemochromatosis
132
6.1 Introduction
Iron plays important roles in both pathogen virulence and host antimicrobial resistance
(Drakesmith and Prentice 2012). Consequently, disturbances in iron metabolism may lead
to changes in host susceptibility to infection, with both primary (Ashrafian 2003) and
secondary (Gangaidzo et al. 2001) iron overload, predisposing an individual to
salmonellosis, tuberculosis and other infections. Conversely, iron deficiency is associated
with relative resistance to infection (Murray et al. 1978).
Synthesis of the key iron regulatory hormone; hepcidin, is up-regulated with iron loading
(Pigeon et al. 2001) and inflammation and down-regulated with anaemia, hypoxia and
increased erythropoiesis (Nicolas et al. 2002). However, in many pathological states,
hepcidin synthesis can be regulated by various synergistic or antagonistic signals. In iron-
loading anaemias such as, thalassaemia and sideroblastic anaemias there is a strong but
inefficient erythropoietic response (Piperno 1998) with the development of anaemia and
hypoxia (Nemeth and Ganz 2006); synergistic signals for the down-regulation of hepcidin
(Nicolas et al. 2002). Conversely, in the anaemia of chronic disease, anaemia occurs
concurrently with inflammation; a condition often seen in autoimmune diseases, chronic
infectious diseases and cancer. In the anaemia of chronic disease, the excessive
production of certain cytokines, in particular IL-6, IL-1β, TNFα and INFγ, resulting in iron
being sequestered by reticuloendothelial macrophages and hepatocytes due to impaired
iron mobilisation (Weiss and Goodnough 2005).
Hepcidin is an antimicrobial peptide of the β-defensin family found in urine (Park et al.
2001) and blood (Krause et al. 2000) and its increased synthesis in response to
lipopolysaccharide (LPS) induced inflammation is mediated via the inflammatory cytokine
IL-6 (Ganz 2003). Although the up-regulation of hepcidin by iron requires the action of
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HFE, TFR2 and HJV, inflammation regulates hepcidin expression via the IL-6 / Janus
kinase 2 (JAK2) -signal transducer and activator of transcription 3 (STAT3) pathway. IL-6
binding to its receptor activates JAK2, which in turn phosphorylates the transcription factor,
STAT3. Phosphorylated STAT3 translocates to the nucleus and binds to the STAT3-
binding site in the proximal region of the hepcidin promoter and upregulates hepcidin gene
expression (Wrighting and Andrews 2006; Pietrangelo et al. 2007; Verga Falzacappa et al.
2007). There is also evidence of crosstalk between this pathway and that of the BMP-
SMAD signaling pathway, as a functional SMAD4 (Wang et al. 2005) and a SMAD-binding
site on the hepcidin promoter has been shown to be a requirement for IL-6-mediated
hepcidin expression (Huang et al. 2009).
The aim of this study was to examine the roles of HFE and TFR2 in the inflammatory
regulation of hepcidin in Hfe knockout (Hfe-/-), Tfr2 Y245X knock-in (Tfr2mut) and HfexTfr2
double mutant (Hfe-/-xTfr2mut) mice and how inflammation together with the dysregulation
of hepcidin synthesis in HH affects iron status.
6.2 Methods
6.2.1 Animals Animal models were generated and raised as previously described (Materials and
methods 2.2.1). Mice were injected with either 0.25mg/kg LPS from Escherichia coli
055:B5 (Sigma-Aldrich)(Yeh et al. 2004) in isotonic saline or 250 µL of saline via
intraperitoneal injection.
6.2.2 Tissue collection Liver tissue was collected 4-18 h after LPS or saline injection as described in Materials
and methods 2.2.1.
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6.2.3 Plasma iron parameters Blood was collected by cardiac puncture according to the method previously described
(Materials and Methods 2.2.1). Plasma iron, transferrin saturation and non-transferrin
bound iron assays were conducted according to the methods described in Materials and
Methods 2.2.3-5.
6.2.4 Gene expression Measurement of liver gene expression was performed by RT-PCR as described in
Materials and methods 2.2.6-11.
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6.3 Results
6.3.1 Effect of LPS with time on hepcidin expression Liver tissue from wild-type mice was collected at 0, 1, 4, and 18 h post LPS injection to
determine the time point at which hepcidin gene expression was most influenced by LPS
stimulation. One h after administration of 0.25 mg/kg LPS (Yeh et al. 2004), hepatic
Hamp1 mRNA expression was increased two-fold and after 4 h Hamp1 levels were 3-fold
higher compared to base-line levels. After 18 h, Hamp1 expression was decreased to
below initial levels (Fig. 6.1). Based on this data, all subsequent analyses were conducted
on liver and blood samples collected 4 h post-LPS administration.
0 1 4 18
0
20
40
60
80
Hamp1
/β-A
ctin
mR
NA
co
py
nu
mb
er
Figure 6.1: LPS time course. Liver Hamp1 mRNA expression was determined by real-time qPCR in wild-type (WT) mice at 0, 1, 4, 18 h after injection with LPS.
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6.3.2 Hepatic expression of inflammatory genes The hepatic inflammatory response induced by LPS administration was examined by
measuring the inflammatory cytokines IL-1α, IL-6, IL-17, and TNFα gene expression by
RT-PCR. Expression of IL-1α was similar in HH and wild-type untreated mice but was
approximately 30% higher in iron-loaded wild-type mice (p<0.05; Fig. 6.2A). IL-1α
expression was increased by more than 2-fold in all LPS treated mice compared with
untreated mice (p<0.01; hatched vs. non-hatched bars). LPS treated Hfe-/-xTfr2mut mice
had significantly (p<0.05) increased IL-1α expression compared with LPS treated wild-type
and iron-loaded wild-type mice.
Hepatic expression of IL-6 was similar in LPS treated HH (Hfe-/-xTfr2mut, Tfr2mut, Hfe-/-) and
iron-loaded wild-type mice and was increased by approximately 23-fold compared with
untreated mice (p<0.01). In LPS-treated mice, IL-6 expression in HH and iron-loaded wild-
type mice was more than 2-fold higher than in non-iron-loaded mice (p<0.05; Fig. 6.2B,
hatched bars vs. non-hatched bars).
LPS treatment resulted in 14-, 9- and 35-fold increases in IL-17a mRNA expression in Hfe-
/-, Tfr2mut and Hfe-/-xTfr2mut mice, respectively (p<0.05; Fig. 6.2C, hatched bars vs. non-
hatched bars). No change in IL-17a expression was observed in LPS treated non-iron
loaded wild-type mice.
Treatment with LPS resulted in an 8-fold increase in TNFα mRNA expression in Hfe-/-
xTfr2mut mice and approximately a 7-fold increase in Hfe-/- and Tfr2mut mice when
compared with untreated mice (p<0.05; Fig. 6.2D, hatched bars vs. non-hatched bars).
LPS treated Hfe-/-xTfr2mut mice were 30% higher than LPS treated Hfe-/- and Tfr2mut mice,
and 2-fold higher than LPS treated wild-type mice (p<0.05; Fig. 6.2D).
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Figure 6.2: Liver expression of inflammatory cytokine genes: LPS vs. saline treated mice. mRNA expression was determined by real-time qPCR. IL-1α (A), IL-6 (B), IL-17 and (C) and TNFα (D) mRNA expression was measured in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe knockout (Hfe-/-), Tfr2 mutant (Tfr2mut) and Hfe-/-xTfr2mut mice after intraperitoneal injection of saline (non-hatched bars) or LPS (hatched bars). Results are expressed as mean ± SEM (n=4-8). Saline: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. LPS: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. *, p<0.05 LPS vs saline.
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6.3.3 Plasma iron parameters Plasma iron concentration in LPS and saline treated mice was higher in HH and iron-
loaded wild-type mice compared with non-iron-loaded wild-type mice (p<0.05; Fig. 6.3A).
Administration of LPS resulted in a 30% reduction of plasma iron levels in non-iron-loaded
and iron-loaded wild-type, and Hfe-/- mice compared with their saline counterparts (p<0.05;
Fig. 6.3A, hatched vs. non-hatched bars) while plasma iron levels in Tfr2mut and Hfe-/-
xTfr2mut mice were unchanged. Similarly, transferrin saturation in LPS and saline treated
mice was higher in all HH and iron-loaded wild-type mice compared with non-iron-loaded
wild-type mice (Fig. 6.3B). However, LPS treatment reduced transferrin saturation in wild-
type and Tfr2mut mice only (p<0.05; Fig. 6.3B, hatched vs. non-hatched bars). Plasma
NTBI concentration in LPS and saline treated mice was elevated in all HH and iron-loaded
wild-type mice (p<0.05; Fig. 6.3C). LPS treatment significantly (p<0.05) reduced plasma
NTBI levels in all types of mice compared with their saline treated counterparts, with a
reduction of almost 40% observed in Hfe-/-xTfr2mut mice (Fig. 6.3C, hatched vs. non-
hatched bars).
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Figure 6.3: Plasma iron parameters: LPS vs. saline treated mice. Plasma iron concentration (A), transferrin saturation (B) and non-transferrin bound iron (NTBI) concentration (C) were measured in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe-/-, Tfr2mut and Hfe-/-xTfr2mut mice after intraperitoneal injection of saline (non-hatched bars) or LPS (hatched bars). Results are expressed as mean ± SEM (n=5-10). Saline: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. LPS: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. *, p<0.05 LPS vs saline.
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6.3.4 Hepatic expression of iron regulatory genes The hepatic expression of the iron regulatory genes Hfe, Tfr2, Bmp6 and Hamp1 were
measured via RT-qPCR. Hfe expression was unchanged in LPS treated Hfe-/-xTfr2mut,
Tfr2mut, and Hfe-/- mice compared with untreated mice, but was increased by more than
20% in LPS treated wild-type and iron-loaded wild-type mice (p<0.05; Fig. 6.4A, hatched
vs. non-hatched bars). Hepatic Tfr2 expression was decreased by more than 60% in LPS
treated Hfe-/-xTfr2mut, Tfr2mut, Hfe-/- and iron-loaded wild-type mice, and decreased by 40%
in non-iron-loaded wild-type mice (p<0.05; Fig. 6.4B, hatched vs. non-hatched bars).
Bmp6 expression was decreased in all LPS treated mice compared with untreated mice
(p<0.05; Fig. 6.4C, hatched vs. non-hatched bars). Bmp6 expression was similar in all LPS
treated HH mice but lower than LPS treated iron-loaded wild-type mice (p<0.05). Hamp1
expression was increased by approximately 80-, 10-, and 6-fold in LPS treated Hfe-/-
xTfr2mut, Tfr2mut and Hfe-/- mice, respectively (p<0.05; Fig. 6.4D, hatched vs. non-hatched
bars). In LPS treated Hfe-/-xTfr2mut mice Hamp1 expression was more than 50% lower than
in LPS treated Tfr2mut and Hfe-/- mice, and more than 70% lower than LPS treated wild-
type mice. In Hfe-/- and Tfr2mut mice, LPS treatment resulted in Hamp1 levels similar to LPS
treated non-iron–loaded wild-type mice but lower than LPS treated iron-loaded wild-type
mice (p<0.05; Fig. 6.4D, hatched bars vs. non-hatched bars).
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Figure 6.4: Liver expression of iron regulatory genes: LPS vs. saline treated mice. mRNA expression was determined by real-time qPCR. Hfe (A), Tfr2 (B), Bmp6 (C), and Hamp1 (D) mRNA expression were measured in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe knockout (Hfe-/-), Tfr2 mutant (Tfr2mut) and Hfe-/-xTfr2mut mice after intraperitoneal injection of saline (non-hatched bars) or LPS (hatched bars). Results are expressed as mean ± SEM (n=4-8). Saline: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. LPS: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. *, p<0.05 saline vs. LPS.
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6.3.5 Hepatic expression of iron transport genes Tfr1 mRNA expression was decreased in all HH and iron-loaded wild-type mice by more
than 40% compared with wild-type mice. Tfr1 gene expression was further decreased by
more than 30-50% in Hfe-/-, Tfr2mut mice and Hfe-/-xTfr2mut mice treated with LPS (p<0.05;
Fig. 6.5A, hatched bars vs. non-hatched bars). Hepatic expression of Zip14 was increased
markedly in all mice after administration of LPS with a 6-8 -fold increase in Zip14 mRNA in
Hfe-/- and Tfr2mut mice and a 5-fold increase in Hfe-/-xTfr2mut mice when compared to
untreated mice (p<0.05; Fig. 6.5B, hatched bars vs. non-hatched bars). Expression of Fpn
was similar in all LPS treated mice and was reduced by more than 70% when compared to
the saline injected mice (p<0.05; Fig. 6.5C, hatched bars vs. non-hatched bars).
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Figure 6.5: Liver expression of iron transport genes in LPS and saline treated mice. mRNA expression was determined by real-time qPCR. Tfr1 (A), Zip14 (B), and Fpn (C) mRNA expression were measured in wild-type (WT), iron-loaded wild-type (WT+Fe), Hfe knockout (Hfe-/-), Tfr2 mutant (Tfr2mut) and Hfe-/-
xTfr2mut mice after intraperitoneal injection of saline (non-hatched bars) or LPS (hatched bars). Results are expressed as mean ± SEM (n=4-8). Saline: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. LPS: a, p<0.05 vs. WT; b, p<0.05 vs. WT+Fe; c, p<0.05 vs. Hfe-/-; d, p<0.05 vs. Tfr2mut. *, p<0.05 saline vs. LPS.
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6.4 Discussion HFE and TFR2 play important roles in the regulation of the iron regulatory hormone
hepcidin, that controls iron absorption, recycling and storage. However, hepcidin is also
regulated by pro-inflammatory cytokines during inflammation and has been implicated in
the anaemia of chronic disease (Andrews 2004). In the current study, administration of
0.25 mg/kg LPS resulted in an increase in liver Hamp1, IL-1α, IL-6, IL-17a and TNFα
expression after four h in both wild-type and HH mice, and that this was associated with
reductions in plasma iron and transferrin saturation levels in Hfe-/-, Tfr2mut and wild-type
mice compared with untreated mice, but were unchanged in LPS treated Hfe-/-xTfr2mut
mice. Furthermore, there were significant reductions in plasma NTBI levels in all LPS
treated groups of mice with approximately a 30% decrease in Hfe-/-xTfr2mut mice. LPS
treatment also decreased hepatic expression of the iron sensors Tfr2 and Bmp6, and
increased Hamp1 in both HH and wild-type mice. Expression of the iron transport genes
Tfr1 and Fpn was decreased with LPS expression, whereas Zip14 expression was
markedly increased in all mice.
In the current study, inflammation was assessed by measuring liver mRNA levels of IL-1α,
IL-6, TNFα and IL-17, all of which were markedly increased in LPS treated HH and wild-
type mice. The administration of bacterial LPS induces inflammation by binding to Toll-like
receptor 4 (TLR4), activating the transcription factor nuclear factor-kappa B (NF-
κB)(Beutler and Poltorak 2001) and inducing the production of the proinflammatory
cytokines including IL-1α, IL-1β, IL-6, IL-17 and TNFα. The activation of NF-κB has been
shown to be stimulated by iron, with iron loading increasing the expression of NF-κB
responsive genes in macrophages (Xiong et al. 2003). LPS-mediated IL-6 production
occurs via two mechanisms, firstly, IL-6 may be stimulated by the increased synthesis of
IL-1β which with or without IL-1α can complex with the IL-1 receptor and IL-1 receptor
accessory protein to stimulate IL-6 synthesis. IL-6 production may also be stimulated by
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the binding of TNFα to its p55 and p75 receptors resulting in increased IL-6 production
(Zetterstrom et al. 1998).
IL-17a is secreted by T helper 17 cells (Miossec et al. 2009) and plays an important role in
regulating tissue inflammation through the induction of proinflammatory cytokines, IL-1β,
IL-6 and TNFα (Aggarwal and Gurney 2002). IL-17a mRNA was measured in this study as
another marker of the inflammation in LPS treated mice. IL-17a and TNFα expression in
Hfe-/-xTfr2mut mice was markedly increased compared with Hfe-/- and Tfr2mut mice and this
may be due to the increased iron burden in these mice accentuating the activation of NF-
κB and in turn IL-17a and TNFα, or it may reflect the early signs of liver injury evident in
Hfe-/-xTfr2mut mice (see Chapter 5)(Delima et al. 2012). IL-17a also activates signal
transducer and activator of transcription 3 (STAT3)(Kim et al. 2012), which then
translocates to the nucleus, binds to the proximal promoter element of the hepcidin gene
(Hamp1), and activates transcription.
Similarly to IL-17a, the cytokine IL-6 promotes the phosphorylation of STAT3 protein,
resulting in enhanced hepcidin expression. In the current study Hfe-/- and Tfr2mut mice
demonstrated the appropriate upregulation of hepcidin (Hamp) in response to IL-6 mice
(Verga Falzacappa et al. 2007). However, this mechanism was impaired in Hfe-/-xTfr2mut
mice as LPS did not upregulate Hamp1 expression to the same extent as in wild-type, Hfe-
/- or Tfr2mut mice. These findings are consistent with the observations by Wallace et al, who
also described a blunted response to inflammation in the absence of both Hfe and Tfr2
(Wallace et al. 2011). It has been suggested that the decreased levels of hepcidin in Hfe-/-
and Tfr2mut mice and even more so in Hfe-/-xTfr2mut (Wallace et al. 2009; Corradini et al.
2011; Delima et al. 2012), is due to impaired BMP-SMAD signaling.
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In the current study the upregulation of Bmp6 mRNA in iron-loaded wild-type mice was the
appropriate response to increased hepatic iron levels (Corradini et al. 2011), whereas this
response was blunted in HH mice despite higher liver iron levels in Hfe-/-xTfr2mut mice than
found in the iron-loaded wild-type mice (Chapter 5, Fig. 3.3D). BMP6 expression was
downregulated in LPS treated HH and wild-type mice. Although the exact mechanism by
which LPS administration causes BMP6 downregulation is not known, it may occur
through the LPS induced downregulation of the BMP6 co-receptor HJV (Krijt et al. 2004). It
has been shown that LPS administration can downregulate HJV expression by two
mechanisms, firstly, through TLR4 signaling, and secondly via TNFα independently of IL-6
(Constante et al. 2007). Bmp6 expression in LPS treated HH mice was down-regulated to
similar extent (approximately 30%) in Hfe-/-xTfr2mut, Hfe, Tfr2 and wild-type mice
suggesting that there was no defect in the inflammatory regulation of BMP6 in HH mice,
but that the lower levels of Bmp6 in LPS treated HH mice compared with iron-loaded wild-
type mice was merely due to the lower basal level of the untreated HH mice. This is in turn
reflected in the Hamp1 expression in Hfe-/-xTfr2mut mice although able to upregulate
Hamp1 levels after LPS treatment, were not able to do so to the same extent as Hfe, Tfr2
and wild-type mice, resulting in an impaired expression of Hamp1 in Hfe-/-xTfr2mut mice.
In the current study, LPS administration decreased plasma iron levels in Hfe-/- and wild-
type mice. The reduction in plasma iron levels after LPS administration is part of the
physiological response to infectious stimuli. LPS administration upregulated the iron
regulatory hormone hepcidin. Increased hepcidin levels result in a decrease in the levels of
the iron exporter, ferroportin (Nemeth et al. 2004), resulting in decreased iron release from
hepatocytes and reticuloendothelial macrophages, ultimately leading to a decrease in
plasma iron levels. Plasma iron levels were unaltered in Hfe-/-xTfr2mut and Tfr2 mice after
LPS administration despite upregulation of hepcidin, this may be due to the extremely low
basal levels of hepcidin in these mice, that even when upregulated by LPS is unable to
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change ferroportin expression to the extent that altered plasma iron levels within the time
frame of this experiment.
LPS administration resulted in dramatic decrease in plasma NTBI levels in both HH and
wild-type mice, suggesting that LPS stimulated the removal of NTBI from the plasma,
possibly mediated by the NTBI transporter Zip14 (Liuzzi et al. 2006). In the current study
Zip14 expression was dramatically upregulated by LPS administration, this is in agreement
with previous studies which reported that liver Zip14 expression was regulated strongly by
IL-6 (Liuzzi et al. 2005). Furthermore liver Zip14 expression was increased in Hfe-/-xTfr2mut
and Tfr2mut mice suggesting a synergistic effect of liver iron loading and inflammation on
Zip14 expression. Also Fpn gene expression was found to be dramatically downregulated
in HH and wild-type mice by LPS treatment. Fpn is not regulated by iron at a
transcriptional level but by a post-translational level by hepcidin. However, the
downregulation of Fpn gene expression by LPS has also previously been shown to involve
the proinflammatory cytokines IL-1 and IL-6 (Liu et al. 2005), in a manner independent of
TNFα (Yang et al. 2002).
The anaemia of chronic disease is typically characterised by immune cell activation and an
inflammatory cytokine response via IL-6, and to lesser extent IL-1 and TNFα (Weiss and
Goodnough 2005), ultimately resulting in dysregulated iron homeostasis and impaired
erythropoiesis. This is supported by findings in the current study where LPS induced
inflammation resulted in increased expression of the iron importer, ZIP14 resulting in
decreased plasma levels of iron and NTBI and a concurrent decrease in the mRNA
expression of the iron exporter Fpn. Inflammation also resulted in an increase in hepcidin
mRNA, though this effect was diminished in HH mice. The concurrent increase in iron
importer levels and decrease in iron exporter levels, results in an iron sequestration
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phenotype evident in the anaemia of chronic disease reducing the bioavailability of iron for
erythropoiesis.
In patients with HH, the immune response to infection is altered, with increased
susceptibility to pathogens such as Vibrio fulnificus (Bullen et al. 1991) as well as
concurrent resistance to macrophage-resident pathogens (Paradkar et al. 2008). The
increased susceptibility to certain pathogens is suggested to be due to attenuated
inflammatory cytokine production in HH by iron deficient macrophages (Wang et al. 2008).
Conversely, it has been proposed that the increased resistance to bacterial infection in
Hfe-/- mice, is due to the enhanced production of the iron sequestering (Flo et al. 2004)
enterochelin-binding peptide, lipocalin-2, in response to HFE disruption (Nairz et al. 2009).
As evident in this study, mutations in iron regulatory genes HFE and TFR2 has a
significant effect on the immune response to bacterial pathogens. Iron plays important
roles in both pathogen virulence and host antimicrobial resistance (Drakesmith and
Prentice 2012). Consequently, disturbances in iron metabolism may lead to changes in
host susceptibility to infection, either via iron sequestration, as in the anaemia of chronic
disease, or by the attenuation of the innate immune response.
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Chapter 7
General discussion
150
In recent years significant advances have been made in the understanding of hereditary
haemochromatosis (HH), and iron biology, in general. Medical and biological research has
been focused on several areas including firstly, the cellular and molecular mechanisms of
iron metabolism including the regulation of dietary iron absorption, transport, and storage;
secondly, the pathophysiological mechanisms of chronic iron overload; and finally, the
genetic basis of HH, diagnosis, and clinical management of iron overload diseases. The
current study addresses these areas using mouse models HH and a dietary iron overload
mouse model to examine iron transport and iron regulatory mechanisms (Chapter 3, 4, 6)
and the pathophysiological mechanisms of iron overload and iron-induced liver injury
(Chapter 5, 6).
Major breakthroughs in the understanding of HH occurred in 1996 with the discovery of the
haemochromatosis gene, HFE (Feder et al. 1996), and the development of the first HFE
knockout mouse in 1998 (Zhou et al. 1998) Further advances occurred in 2000, with the
discovery of another form of HH, caused by mutation of the TFR2 gene (Camaschella et
al. 2000) and the subsequent development of a TFR2 mutant mouse (Fleming et al. 2002).
Currently it is thought, that HFE and TFR2, form a complex on the cell surface of
hepatocytes to sense plasma transferrin saturation (Gao et al. 2009), whilst BMP6 through
its co-receptor HJV (Xia et al. 2008) and its negative regulator matriptase (Folgueras et al.
2008), sense liver iron levels (Kautz et al. 2008). HFE/TFR2 and BMP6/HJV, possibly as a
multi-protein membrane complex (D'Alessio et al. 2012), signal via the SMAD pathway to
modulate dietary iron absorption and cellular iron release, through the regulation of the
iron regulatory hormone, hepcidin.
In Chapter 3, it was shown that mice with disruption in both HFE and TFR2 (Hfe-/-xTfr2mut)
develop a more severe iron loading phenotype than mice with disruption of either HFE
(Hfe-/-) or TFR2 (Tfr2mut) alone. Haematological parameters (RBC, Hb, and Hct) were all
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higher in Hfe-/-xTfr2mut and Tfr2mut mice than in Hfe-/- mice. Iron status (plasma iron,
transferrin saturation and NTBI concentration) and liver iron content were all increased in
Hfe-/-xTfr2mut mice, and significantly higher than in Tfr2mut and Hfe-/- mice, indicative of a
more severe iron loading phenotype. These findings are consistent with the evidence
obtained in other models of HFE and TFR2 disruption (Wallace et al. 2009; Corradini et al.
2011). Hfe-/-xTfr2mut mice had elevated liver Bmp6 levels consistent with increased liver
iron content, however disruption of Hfe and Tfr2 expression resulted in ineffective p-Smad
1,5,8 signalling leading to reduced liver Hamp1.
The disruption of HFE and TFR2 in the Hfe-/-xTfr2mut mouse resulted in a form of HH
comparable to HH type 2, a disorder that is characterised by a severe, early onset iron
overload, with increased iron absorption and liver iron accumulation that is greater than
HH type 1 (HFE mutations) or type 3 (TFR2 mutations), leading to severe organ
impairment with hypogonadism and cardiac involvement prominent features of the clinical
syndrome. HH type 2 occurs as a result of mutations in HJV or the gene encoding the iron
regulatory hepcidin, known as HAMP. The observation that the combined disruption of
HFE and TFR2 resulted in a more severe phenotype than disruption of either HFE or
TFR2 alone suggests a model of iron-dependent regulation of hepcidin where both HFE
and TFR2 act as plasma iron sensors via parallel and possibly converging signalling
pathways that is as important as BMP6/HJV in the regulation of hepcidin.
Decreased hepcidin expression results in enhanced release of iron from intestinal
enterocytes and macrophages, which saturates plasma transferrin leading to the increased
presence of plasma NTBI as observed in Chapter 3 (Fig 3.1). As previously mentioned, the
predominant form of NTBI in HH is iron-citrate (Grootveld et al. 1989), however the uptake
and tissue distribution of NTBI in HH is still poorly understood. The kinetics of hepatic
NTBI transport have been predominantly studied in cell lines and isolated cells, utilising
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the human hepatoma Huh7 cell line (Trinder and Morgan 1998), and isolated rat (Baker et
al. 1998) and mouse (Chua et al. 2004) hepatocytes. These studies have provided
invaluable information regarding the mechanisms of NTBI uptake. The use of isolated cells
to the study of NTBI transport however, neglects the importance of complex interactions
between the circulation and various cells and tissues in vivo. In Chapter 4, in vivo NTBI
transport was determined in the mouse models of HH. It was shown that NTBI was cleared
rapidly from the circulation in all mouse models of HH, with most of the NTBI taken up by
the liver and to a lesser degree by the kidneys, pancreas and heart. This Chapter
describes for the first time increased in vivo tissue NTBI uptake in mouse models of HH,
and the positive correlation between NTBI uptake and both plasma NTBI levels and iron
content in the liver, kidney, pancreas and heart. This data suggest that NTBI uptake is
likely to contribute to excessive tissue iron deposition in HH. The only previously
documented studies of in vivo NTBI transport used wild-type rats with iron-saturated
transferrin and hypotransferrinaemic mice. In that study, NTBI was also shown to be
cleared rapidly from the plasma in rats with iron-saturated transferrin and
hypotransferrinaemic mice, with the majority of radiolabeled iron citrate taken up by the
liver (Craven et al. 1987).
Free iron is redox-active and can generate ROS via the Fenton and Haber-Weiss
reactions, leading to lipid peroxidation, lysosomal fragility and mitochondrial and DNA
damage (Britton et al. 2002). Although other models of combined HFE and TFR2
disruption display a similar iron overload phenotype to that of the Hfe-/-xTfr2mut mouse
(Wallace et al. 2009; Corradini et al. 2011), to date, there has been no report of iron-
induced liver injury in murine models of HH (Subramaniam et al. 2012), in stark contrast to
the well described injury in the human disease (Deugnier et al. 1992). In Chapter 5, the
first HH mouse model of iron-induced liver injury, the Hfe-/-xTfr2mut mouse, is described. In
association with increased plasma iron levels and elevated hepatic periportal iron
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deposition (Chapter 3), there was significant elevation of plasma ALT activity in Hfe-/-
xTfr2mut mice. There was also mild hepatic inflammatory cell infiltration with scattered foci
of CD45+ leukocytes colocalised predominately with ferritin in portal regions of the liver and
evidence of hepatocyte sideronecrosis in Hfe-/-xTfr2mut mice. Elevated hydroxyproline
levels, and Sirius red and Trichrome staining demonstrated marked portal tract collagen
deposition and portal bridging in Hfe-/-xTfr2mut mice, strongly supporting the presence of
liver fibrosis in areas of high iron accumulation. Histological evidence of a more
pronounced liver damage in Hfe-/-xTfr2mut mice was corroborated by increased expression
of TNFα, and decreased SOD activity and enhanced lipid peroxidation in the liver,
indicating elevated hepatic oxidative stress.
As previously described the genetic background of HH mice may contribute to the iron-
induced liver injury in murine HH. The AKR mouse strain is often described as an iron-
loading strain, with liver iron content, transferrin saturation (McLachlan et al. 2011), and in
turn lipid peroxidation product malondialdehyde levels (Gerhard et al. 2002) twice that of
the commonly used C57BL/6 strain, such that the liver injury described in Chapter 5 may
result from higher basal levels of iron in the AKR genetic strain than observed in
comparable models of HFE and TFR2 disruption on C57BL/6 (Wallace et al. 2009) and
FVB (Corradini et al. 2011) genetic backgrounds. Interestingly, AKR mice also have higher
levels of liver TBARS than the higher-iron-loading mouse strain, CBA (Sverko et al. 2002),
suggesting that iron levels alone may not be the only factor influencing liver injury in the
Hfe-/-xTfr2mut mouse. In fact, studies in human HH have shown a correlation between
genetic dimorphisms in the antioxidant enzymes SOD2 and myeloperoxidase and
increased rates of cirrhosis and hepatocellular carcinoma, suggesting that iron-induced
liver injury may be influenced by an inability to effectively ameliorate the effects of ROS,
this may also influence the degree of iron-induced injury evident in the Hfe-/-xTfr2mut
mouse.
154
Although HH is not typically considered an inflammatory disease, evidence of mild
inflammation (Stal et al. 1995; Bridle et al. 2003) and periportal sideronecrosis (Deugnier
et al. 1992) has been observed in human HH liver and, as mentioned previously, in the
Hfe-/-xTfr2mut mouse (Chapter 5). In addition, inflammatory stimuli have been shown to
have a significant effect on the regulation of hepcidin (Drakesmith and Prentice 2012) and
a number of iron transporters causing anaemia of inflammation where iron required for
erythropoiesis is sequestered in the liver. In Chapter 6, the administration of LPS to HH
mice induced inflammation resulting in decreased plasma iron and NTBI levels and a
concurrent increase in the mRNA expression of liver iron importer Zip14 and decrease in
the iron exporter Fpn. Inflammation also increased hepcidin mRNA, though this effect was
diminished in HH mice. Increased hepcidin levels would result in decreased FPN protein
expression and hepatic iron export. The concurrent increase in iron importer levels and
decrease in iron exporter levels, results in liver iron retention evident in the anaemia of
inflammation, reducing the bioavailability of iron for erythropoiesis. In chronic inflammatory
conditions, with iron sequesteration in the liver, iron overload may occur over time and
cause liver injury as described in Chapter 5.
In conclusion, the disruption of both HFE and TFR2 results in an inability to sense plasma
iron levels leading to decreased synthesis of the hepatic iron regulator hepcidin, resulting
in elevated iron levels, and unmitigated iron absorption. Excess iron saturated circulating
transferrin, resulting in the presence of NTBI. Plasma NTBI was rapidly removed from the
circulation and predominantly deposited in the liver, kidney, pancreas and heart with NTBI
uptake positively correlated with both plasma NTBI and tissue iron content. The deposition
of excess iron in the liver likely contributes to the iron-induced liver injury and fibrosis in
Hfe-/-xTfr2mut mice, with liver NTBI uptake positively correlating with collagen deposition.
Systemic inflammation may also exacerbate the iron sequestration contributing further to
liver iron overload and iron-induced injury. The use of the Hfe-/-xTfr2mut mouse model of HH
155
provides not only greater insight into the mechanisms of iron-induced liver injury, but also
a model for the screening of new therapeutics such as modifiers of the hepcidin pathway
and inhibitors of inflammatory pathways, to treat iron overload disorders and the anaemia
of chronic disease.
7.1 Future directions
7.1.1 The role of HFE and TFR2 and erythropoiesis An interesting finding from this study was the observation of increased haematological
parameters in conjunction with splenomegaly, in Tfr2mut and Hfe-/-xTfr2mut mice indicating a
role for TFR2 in erythropoiesis. Apart from stimulating erythropoiesis by increasing
available iron through hepcidin inhibition, TFR2 is proposed to play a role in early
erythropoiesis and erythropoietin sensitivity. However, the exact mechanism by which this
occurs is not fully understood and deserves further investigation. Experiments should
focus on measurement of serum erythropoietin and examination of the bone marrow in
Tfr2mut and Hfe-/-xTfr2mut mouse to confirm increased erythropoiesis and identify any
abnormalities in erythropoietic function.
7.1.2 NTBI Transporters Though much is known about the clearance, uptake, and distribution of NTBI, the
molecular mechanisms by which uptake occurs are yet to be fully elucidated. DMT1 and
ZIP14 have been proposed as candidate NTBI transporters, with studies showing
increased NTBI uptake in overexpressing cell lines (Liuzzi et al. 2006; Shindo et al. 2006)
and HFE knockout hepatocytes (Chua et al. 2004). However, the hepatic expression and
localisation of DMT1 and ZIP14 in murine HH has been hampered by the lack of suitable
antibodies. Studies in mice with deletion of hepatic DMT1 are still capable of accumulating
hepatic iron (Gunshin et al. 2005), in addition, the knock-out of ZIP14 in mice did not result
156
in decreased iron levels (Nam et al. 2013). These results suggest that DMT1 and ZIP14
may not play a major role in NTBI uptake and that other NTBI transporters exist. Future
experiments should also examine the expression and localisation of ZIP8 and L-type
calcium channels in the pancreas and heart and lipocalin in the kidney, to determine if their
expression is regulated by iron levels in murine HH.
7.1.3 Iron-induction of liver injury Studies in the Hfe-/-xTfr2mut mouse have shown a strong positive correlation between liver
iron content, plasma NTBI concentration and liver injury. Also, in unpublished studies from
this laboratory, dietary iron-loading and increasing age of Hfe-/-xTfr2mut mice exacerbated
liver injury. However, to make a definitive statement on whether iron was indeed the
causative agent of liver injury a study should be undertaken in which Hfe-/-xTfr2mut mice are
raised on an iron-deficient diet, to confirm whether liver injury in the Hfe-/-xTfr2mut mouse is
reduced in the absence of iron. In addition, the mechanisms by which iron-induced
oxidative stress and inflammation contribute to liver injury should be examined by
quantifying and delineating the effects of ROS and by examining how lipid peroxidation
products affect mitochondrial function and in turn cell death.
157
Chapter 8
Bibliography
158
Abboud, S. and D. J. Haile (2000). "A novel mammalian iron-regulated protein involved in intracellular iron metabolism." J Biol Chem 275(26): 19906-19912. Adams, P. C. (2001). "Is there a threshold of hepatic iron concentration that leads to cirrhosis in C282Y hemochromatosis?" Am J Gastroenterol 96(2): 567-569. Aggarwal, S. and A. L. Gurney (2002). "IL-17: prototype member of an emerging cytokine family." J Leukoc Biol 71(1): 1-8. Ahmad, K. A., J. R. Ahmann, M. C. Migas, A. Waheed, R. S. Britton, B. R. Bacon, W. S. Sly and R. E. Fleming (2002). "Decreased liver hepcidin expression in the Hfe knockout mouse." Blood Cells Mol Dis 29(3): 361-366. Ajioka, R. S., J. E. Levy, N. C. Andrews and J. P. Kushner (2002). "Regulation of iron absorption in Hfe mutant mice." Blood 100(4): 1465-1469. Andersen, R. V., A. Tybjaerg-Hansen, M. Appleyard, H. Birgens and B. G. Nordestgaard (2004). "Hemochromatosis mutations in the general population: iron overload progression rate." Blood 103(8): 2914-2919. Anderson, G. J., L. W. Powell and J. W. Halliday (1994). "The endocytosis of transferrin by rat intestinal epithelial cells." Gastroenterology 106(2): 414-422. Andrews, N. C. (1999). "Disorders of iron metabolism." N Engl J Med 341(26): 1986-1995. Andrews, N. C. (2004). "Anemia of inflammation: the cytokine-hepcidin link." J Clin Invest 113(9): 1251-1253. Andriopoulos, B., Jr., E. Corradini, Y. Xia, S. A. Faasse, S. Chen, L. Grgurevic, M. D. Knutson, A. Pietrangelo, S. Vukicevic, H. Y. Lin and J. L. Babitt (2009). "BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism." Nat Genet 41(4): 482-487. Arosio, P. and S. Levi (2010). "Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage." Biochim Biophys Acta 1800(8): 783-792. Arredondo, M., P. Munoz, C. V. Mura and M. T. Nunez (2003). "DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells." Am J Physiol Cell Physiol 284(6): C1525-1530. Aruoma, O. I., A. Bomford, R. J. Polson and B. Halliwell (1988). "Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy." Blood 72(4): 1416-1419. Aruoma, O. I., H. Kaur and B. Halliwell (1991). "Oxygen free radicals and human diseases." J R Soc Health 111(5): 172-177. Ashrafian, H. (2003). "Hepcidin: the missing link between hemochromatosis and infections." Infect Immun 71(12): 6693-6700. Babitt, J. L., F. W. Huang, D. M. Wrighting, Y. Xia, Y. Sidis, T. A. Samad, J. A. Campagna, R. T. Chung, A. L. Schneyer, C. J. Woolf, N. C. Andrews and H. Y. Lin (2006). "Bone
159
morphogenetic protein signaling by hemojuvelin regulates hepcidin expression." Nat Genet 38(5): 531-539. Bacon, B. R., R. O'Neill and C. H. Park (1986). "Iron-induced peroxidative injury to isolated rat hepatic mitochondria." J Free Radic Biol Med 2(5-6): 339-347. Bacon, B. R. and R. S. Britton (1990). "The pathology of hepatic iron overload: a free radical--mediated process?" Hepatology 11(1): 127-137. Bacon, B. R., R. O'Neill and R. S. Britton (1993). "Hepatic mitochondrial energy production in rats with chronic iron overload." Gastroenterology 105(4): 1134-1140. Bacon, B. R., J. K. Olynyk, E. M. Brunt, R. S. Britton and R. K. Wolff (1999). "HFE genotype in patients with hemochromatosis and other liver diseases." Ann Intern Med 130(12): 953-962. Bahram, S., S. Gilfillan, L. C. Kuhn, R. Moret, J. B. Schulze, A. Lebeau and K. Schumann (1999). "Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism." Proc Natl Acad Sci U S A 96(23): 13312-13317. Baker, E., S. M. Baker and E. H. Morgan (1998). "Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture." Biochim Biophys Acta 1380(1): 21-30. Barisani, D., C. L. Berg, M. Wessling-Resnick and J. L. Gollan (1995). "Evidence for a low Km transporter for non-transferrin-bound iron in isolated rat hepatocytes." Am J Physiol 269(4 Pt 1): G570-576. Barton, J. C., L. F. Bertoli and B. E. Rothenberg (2000). "Peripheral blood erythrocyte parameters in hemochromatosis: evidence for increased erythrocyte hemoglobin content." J Lab Clin Med 135(1): 96-104. Beker Aydemir, T., S. M. Chang, G. J. Guthrie, A. B. Maki, M. S. Ryu, A. Karabiyik and R. J. Cousins (2012). "Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia)." PLoS One 7(10): e48679. Besson-Fournier, C., C. Latour, L. Kautz, J. Bertrand, T. Ganz, M. P. Roth and H. Coppin (2012). "Induction of activin B by inflammatory stimuli up-regulates expression of the iron-regulatory peptide hepcidin through Smad1/5/8 signaling." Blood 120(2): 431-439. Beutler, B. and A. Poltorak (2001). "The sole gateway to endotoxin response: how LPS was identified as Tlr4, and its role in innate immunity." Drug Metab Dispos 29(4 Pt 2): 474-478. Blight, G. D. and E. H. Morgan (1983). "Ferritin and iron uptake by reticulocytes." Br J Haematol 55(1): 59-71. Brasse-Lagnel, C., Z. Karim, P. Letteron, S. Bekri, A. Bado and C. Beaumont (2011). "Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation." Gastroenterology 140(4): 1261-1271 e1261.
160
Breuer, W., C. Hershko and Z. I. Cabantchik (2000). "The importance of non-transferrin bound iron in disorders of iron metabolism." Transfus Sci 23(3): 185-192. Bridle, K. R., D. M. Frazer, S. J. Wilkins, J. L. Dixon, D. M. Purdie, D. H. Crawford, V. N. Subramaniam, L. W. Powell, G. J. Anderson and G. A. Ramm (2003). "Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis." Lancet 361(9358): 669-673. Bridle, K. R., D. H. Crawford, L. M. Fletcher, J. L. Smith, L. W. Powell and G. A. Ramm (2003). "Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis." J Hepatol 38(4): 426-433. Brissot, P., T. L. Wright, W. L. Ma and R. A. Weisiger (1985). "Efficient clearance of non-transferrin-bound iron by rat liver. Implications for hepatic iron loading in iron overload states." J Clin Invest 76(4): 1463-1470. Brissot, P., D. Guyader, O. Loreal, F. Laine, A. Guillygomarc'h, R. Moirand and Y. Deugnier (2000). "Clinical aspects of hemochromatosis." Transfus Sci 23(3): 193-200. Britton, R. S., G. A. Ramm, J. Olynyk, R. Singh, R. O'Neill and B. R. Bacon (1994). "Pathophysiology of iron toxicity." Adv Exp Med Biol 356: 239-253. Britton, R. S., K. L. Leicester and B. R. Bacon (2002). "Iron toxicity and chelation therapy." Int J Hematol 76(3): 219-228. Brown, K. E., M. T. Kinter, T. D. Oberley, M. L. Freeman, H. F. Frierson, L. A. Ridnour, Y. Tao, L. W. Oberley and D. R. Spitz (1998). "Enhanced gamma-glutamyl transpeptidase expression and selective loss of CuZn superoxide dismutase in hepatic iron overload." Free Radic Biol Med 24(4): 545-555. Brunt, E. M. (2005). "Pathology of hepatic iron overload." Semin Liver Dis 25(4): 392-401. Bullen, J. J., P. B. Spalding, C. G. Ward and J. M. Gutteridge (1991). "Hemochromatosis, iron and septicemia caused by Vibrio vulnificus." Arch Intern Med 151(8): 1606-1609. Camaschella, C., A. Roetto, A. Cali, M. De Gobbi, G. Garozzo, M. Carella, N. Majorano, A. Totaro and P. Gasparini (2000). "The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22." Nat Genet 25(1): 14-15. Camaschella, C., A. Roetto and M. De Gobbi (2002). "Juvenile hemochromatosis." Semin Hematol 39(4): 242-248. Camaschella, C. and E. Poggiali (2009). "Rare types of genetic hemochromatosis." Acta Haematol 122(2-3): 140-145. Camaschella, C., Roetto, A., Gobbi, M. (2002). "Juvenile hemochromatosis." Seminars in Hematology 39: 242-248. Canonne-Hergaux, F. and P. Gros (2002). "Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice." Kidney Int 62(1): 147-156.
161
Canonne-Hergaux, F., A. Donovan, C. Delaby, H. J. Wang and P. Gros (2006). "Comparative studies of duodenal and macrophage ferroportin proteins." Am J Physiol Gastrointest Liver Physiol 290(1): G156-163. Carlson, H., A. S. Zhang, W. H. Fleming and C. A. Enns (2005). "The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells." Blood 105(6): 2564-2570. Carpenter, C. E., and Mahoney, A.W. (1992). "Contributions of heme and non-heme iron to human nutrition." Critical Reviews in Food Science and Nutrition 31: 333-367. Casanovas, G., K. Mleczko-Sanecka, S. Altamura, M. W. Hentze and M. U. Muckenthaler (2009). "Bone morphogenetic protein (BMP)-responsive elements located in the proximal and distal hepcidin promoter are critical for its response to HJV/BMP/SMAD." J Mol Med (Berl) 87(5): 471-480. Casanovas, G., D. W. Swinkels, S. Altamura, K. Schwarz, C. M. Laarakkers, H. J. Gross, M. Wiesneth, H. Heimpel and M. U. Muckenthaler (2011). "Growth differentiation factor 15 in patients with congenital dyserythropoietic anaemia (CDA) type II." J Mol Med (Berl) 89(8): 811-816. Cavill, I. (2002). "Erythropoiesis and iron." Best Pract Res Clin Haematol 15(2): 399-409. Chambers, J. C., W. Zhang, Y. Li, J. Sehmi, M. N. Wass, D. Zabaneh, C. Hoggart, H. Bayele, M. I. McCarthy, L. Peltonen, N. B. Freimer, S. K. Srai, P. H. Maxwell, M. J. Sternberg, A. Ruokonen, G. Abecasis, M. R. Jarvelin, J. Scott, P. Elliott and J. S. Kooner (2009). "Genome-wide association study identifies variants in TMPRSS6 associated with hemoglobin levels." Nat Genet 41(11): 1170-1172. Chaston, T., B. Chung, M. Mascarenhas, J. Marks, B. Patel, S. K. Srai and P. Sharp (2008). "Evidence for differential effects of hepcidin in macrophages and intestinal epithelial cells." Gut 57(3): 374-382. Chen, J. and C. A. Enns (2007). "The Cytoplasmic domain of transferrin receptor 2 dictates its stability and response to holo-transferrin in Hep3B cells." J Biol Chem 282(9): 6201-6209. Chen, J., M. Chloupkova, J. Gao, T. L. Chapman-Arvedson and C. A. Enns (2007). "HFE modulates transferrin receptor 2 levels in hepatoma cells via interactions that differ from transferrin receptor 1-HFE interactions." J Biol Chem 282(51): 36862-36870. Chen, T. T., L. Li, D. H. Chung, C. D. Allen, S. V. Torti, F. M. Torti, J. G. Cyster, C. Y. Chen, F. M. Brodsky, E. C. Niemi, M. C. Nakamura, W. E. Seaman and M. R. Daws (2005). "TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis." J Exp Med 202(7): 955-965. Choi, J. H., Y. Latunde-Dada, A.T. Laftah, and Simpson, R.J. (2008). The role of DcytB in intestinal iron absorption, (abstract). Choi, S. O., Y. S. Cho, H. L. Kim and J. W. Park (2007). "ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBPalpha and STAT-3." Biochem Biophys Res Commun 356(1): 312-317.
162
Chua, A. C., J. K. Olynyk, P. J. Leedman and D. Trinder (2004). "Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis." Blood 104(5): 1519-1525. Chua, A. C., R. M. Graham, D. Trinder and J. K. Olynyk (2007). "The regulation of cellular iron metabolism." Crit Rev Clin Lab Sci 44(5-6): 413-459. Chua, A. C., C. E. Herbison, S. F. Drake, R. M. Graham, J. K. Olynyk and D. Trinder (2008). "The role of Hfe in transferrin-bound iron uptake by hepatocytes." Hepatology 47(5): 1737-1744. Chua, A. C., R. D. Delima, E. H. Morgan, C. E. Herbison, J. E. Tirnitz-Parker, R. M. Graham, R. E. Fleming, R. S. Britton, B. R. Bacon, J. K. Olynyk and D. Trinder (2010). "Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis." J Hepatol 52(3): 425-431. Chung, J., Wessling-Resnick, M. (2003). "Molecular mechanisms and regulation of iron transport." Critical Reviews in Clinical Laboratory Sciences 40: 151-182. Conrad, M. E., Benjamin, B.I., Williams, H.L., Foy, A.L. (1967). "Human absorption of hemoglobin iron." Gastroenterology 53: 5-10. Constante, M., W. Jiang, D. Wang, V. A. Raymond, M. Bilodeau and M. M. Santos (2006). "Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation." Am J Physiol Gastrointest Liver Physiol 291(2): G229-237. Constante, M., D. Wang, V. A. Raymond, M. Bilodeau and M. M. Santos (2007). "Repression of repulsive guidance molecule C during inflammation is independent of Hfe and involves tumor necrosis factor-alpha." Am J Pathol 170(2): 497-504. Corradini, E., M. Rozier, D. Meynard, A. Odhiambo, H. Y. Lin, Q. Feng, M. C. Migas, R. S. Britton, J. L. Babitt and R. E. Fleming (2011). "Iron regulation of hepcidin despite attenuated Smad1,5,8 signaling in mice without transferrin receptor 2 or Hfe." Gastroenterology. Correnti, C., V. Richardson, A. K. Sia, A. D. Bandaranayake, M. Ruiz, Y. Suryo Rahmanto, Z. Kovacevic, M. C. Clifton, M. A. Holmes, B. K. Kaiser, J. Barasch, K. N. Raymond, D. R. Richardson and R. K. Strong (2012). "Siderocalin/Lcn2/NGAL/24p3 does not drive apoptosis through gentisic acid mediated iron withdrawal in hematopoietic cell lines." PLoS One 7(8): e43696. Craven, C. M., J. Alexander, M. Eldridge, J. P. Kushner, S. Bernstein and J. Kaplan (1987). "Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis." Proc Natl Acad Sci U S A 84(10): 3457-3461. D'Alessio, F., M. W. Hentze and M. U. Muckenthaler (2012). "The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation." J Hepatol 57(5): 1052-1060. De Domenico, I., M. B. Vaughn, L. Li, D. Bagley, G. Musci, D. M. Ward and J. Kaplan (2006). "Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome." Embo J 25(22): 5396-5404.
163
De Domenico, I., D. M. Ward, G. Musci and J. Kaplan (2007). "Evidence for the multimeric structure of ferroportin." Blood 109(5): 2205-2209. De Domenico, I., D. M. Ward, C. Langelier, M. B. Vaughn, E. Nemeth, W. I. Sundquist, T. Ganz, G. Musci and J. Kaplan (2007). "The molecular mechanism of hepcidin-mediated ferroportin down-regulation." Mol Biol Cell 18(7): 2569-2578. De Domenico, I., D. M. Ward, M. C. di Patti, S. Y. Jeong, S. David, G. Musci and J. Kaplan (2007). "Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin." Embo J 26(12): 2823-2831. De Domenico, I., T. Y. Zhang, C. L. Koening, R. W. Branch, N. London, E. Lo, R. A. Daynes, J. P. Kushner, D. Li, D. M. Ward and J. Kaplan (2010). "Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice." J Clin Invest 120(7): 2395-2405. De Domenico, I., E. Lo, B. Yang, T. Korolnek, I. Hamza, D. M. Ward and J. Kaplan (2011). "The role of ubiquitination in hepcidin-independent and hepcidin-dependent degradation of ferroportin." Cell Metab 14(5): 635-646. De Feo, T. M., S. Fargion, L. Duca, B. M. Cesana, L. Boncinelli, P. Lozza, M. D. Cappellini and G. Fiorelli (2001). "Non-transferrin-bound iron in alcohol abusers." Alcohol Clin Exp Res 25(10): 1494-1499. Delima, R. D., A. C. Chua, J. E. Tirnitz-Parker, E. K. Gan, K. D. Croft, R. M. Graham, J. K. Olynyk and D. Trinder (2012). "Disruption of HFE and TFR2 causes iron-induced liver injury in mice." Hepatology. Deugnier, Y. M., O. Loreal, B. Turlin, D. Guyader, H. Jouanolle, R. Moirand, C. Jacquelinet and P. Brissot (1992). "Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations." Gastroenterology 102(6): 2050-2059. Devireddy, L. R., C. Gazin, X. Zhu and M. R. Green (2005). "A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake." Cell 123(7): 1293-1305. Donovan, A., C. A. Lima, J. L. Pinkus, G. S. Pinkus, L. I. Zon, S. Robine and N. C. Andrews (2005). "The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis." Cell Metab 1(3): 191-200. Doolittle, R. L. and G. W. Richter (1981). "Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron." Lab Invest 45(6): 567-574. Drake, S. F., E. H. Morgan, C. E. Herbison, R. Delima, R. M. Graham, A. C. Chua, P. J. Leedman, R. E. Fleming, B. R. Bacon, J. K. Olynyk and D. Trinder (2007). "Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3." Am J Physiol Gastrointest Liver Physiol 292(1): G323-328. Drakesmith, H. and A. M. Prentice (2012). "Hepcidin and the iron-infection axis." Science 338(6108): 768-772.
164
Dupic, F., S. Fruchon, M. Bensaid, N. Borot, M. Radosavljevic, O. Loreal, P. Brissot, S. Gilfillan, S. Bahram, H. Coppin and M. P. Roth (2002). "Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mRNAs between mouse strains." Gastroenterology 122(3): 745-751. el Ghissassi, F., A. Barbin, J. Nair and H. Bartsch (1995). "Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases." Chem Res Toxicol 8(2): 278-283. Elmberg, M., R. Hultcrantz, A. Ekbom, L. Brandt, S. Olsson, R. Olsson, S. Lindgren, L. Loof, P. Stal, S. Wallerstedt, S. Almer, H. Sandberg-Gertzen and J. Askling (2003). "Cancer risk in patients with hereditary hemochromatosis and in their first-degree relatives." Gastroenterology 125(6): 1733-1741. Feder, J. N., A. Gnirke, W. Thomas, Z. Tsuchihashi, D. A. Ruddy, A. Basava, F. Dormishian, R. Domingo, Jr., M. C. Ellis, A. Fullan, L. M. Hinton, N. L. Jones, B. E. Kimmel, G. S. Kronmal, P. Lauer, V. K. Lee, D. B. Loeb, F. A. Mapa, E. McClelland, N. C. Meyer, G. A. Mintier, N. Moeller, T. Moore, E. Morikang, C. E. Prass, L. Quintana, S. M. Starnes, R. C. Schatzman, K. J. Brunke, D. T. Drayna, N. J. Risch, B. R. Bacon and R. K. Wolff (1996). "A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis." Nat Genet 13(4): 399-408. Fernandes, A., G. C. Preza, Y. Phung, I. De Domenico, J. Kaplan, T. Ganz and E. Nemeth (2009). "The molecular basis of hepcidin-resistant hereditary hemochromatosis." Blood 114(2): 437-443. Fielding, J. (1980). Serum iron and iron binding capacity. Methods in Hematology, IRON. J. D. Cook, Churchill Livingstone: 15-43. Fleming, R. E., J. R. Ahmann, M. C. Migas, A. Waheed, H. P. Koeffler, H. Kawabata, R. S. Britton, B. R. Bacon and W. S. Sly (2002). "Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis." Proc Natl Acad Sci USA 99(16): 10653-10658. Flo, T. H., K. D. Smith, S. Sato, D. J. Rodriguez, M. A. Holmes, R. K. Strong, S. Akira and A. Aderem (2004). "Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron." Nature 432(7019): 917-921. Folgueras, A. R., F. M. de Lara, A. M. Pendas, C. Garabaya, F. Rodriguez, A. Astudillo, T. Bernal, R. Cabanillas, C. Lopez-Otin and G. Velasco (2008). "Membrane-bound serine protease matriptase-2 (Tmprss6) is an essential regulator of iron homeostasis." Blood 112(6): 2539-2545. Forejtnikova, H., M. Vieillevoye, Y. Zermati, M. Lambert, R. M. Pellegrino, S. Guihard, M. Gaudry, C. Camaschella, C. Lacombe, A. Roetto, P. Mayeux and F. Verdier (2010). "Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis." Blood 116(24): 5357-5367. Francanzani, A. L., S. Fargion, R. Romano, A. Piperno, P. Arosio, G. Ruggeri, G. Ronchi and G. Fiorelli (1989). "Immunohistochemical evidence for a lack of ferritin in duodenal absorptive epithelial cells in idiopathic hemochromatosis." Gastroenterology 96(4): 1071-1078.
165
Frazer, D. A., Wilkins, S.J., Becker, E.M., Vulpe, C.D., McKie, A.T., Trinder, D. and Anderson, G.J. (2002). "Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats." Gastroenterology 123: 835-844. Gaasch, J. A., W. J. Geldenhuys, P. R. Lockman, D. D. Allen and C. J. Van der Schyf (2007). "Voltage-gated calcium channels provide an alternate route for iron uptake in neuronal cell cultures." Neurochem Res 32(10): 1686-1693. Gangaidzo, I. T., V. M. Moyo, E. Mvundura, G. Aggrey, N. L. Murphree, H. Khumalo, T. Saungweme, I. Kasvosve, Z. A. Gomo, T. Rouault, J. R. Boelaert and V. R. Gordeuk (2001). "Association of pulmonary tuberculosis with increased dietary iron." J Infect Dis 184(7): 936-939. Ganz, T. (2003). "Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation." Blood 102(3): 783-788. Gao, J., N. Zhao, M. D. Knutson and C. A. Enns (2008). "The hereditary hemochromatosis protein, HFE, inhibits iron uptake via down-regulation of Zip14 in HepG2 cells." J Biol Chem 283(31): 21462-21468. Gao, J., J. Chen, M. Kramer, H. Tsukamoto, A. S. Zhang and C. A. Enns (2009). "Interaction of the hereditary hemochromatosis protein HFE with transferrin receptor 2 is required for transferrin-induced hepcidin expression." Cell Metab 9(3): 217-227. Garrick, M. D., S. T. Singleton, F. Vargas, H. C. Kuo, L. Zhao, M. Knopfel, T. Davidson, M. Costa, P. Paradkar, J. A. Roth and L. M. Garrick (2006). "DMT1: which metals does it transport?" Biol Res 39(1): 79-85. Gerhard, G. S., E. J. Kaufmann, X. Wang, K. M. Erikson, J. Abraham, M. Grundy, J. L. Beard and M. J. Chorney (2002). "Genetic differences in hepatic lipid peroxidation potential and iron levels in mice." Mech Ageing Dev 123(2-3): 167-176. Ghio, A. J., E. Nozik-Grayck, J. Turi, I. Jaspers, D. R. Mercatante, R. Kole and C. A. Piantadosi (2003). "Superoxide-dependent iron uptake: a new role for anion exchange protein 2." Am J Respir Cell Mol Biol 29(6): 653-660. Gosriwatana, I., Loreal, O., Lu, S., Brissot, P., Porter, J., Hider, R.C. (1999). "Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin." Analytical Biochemistry 273: 212-220. Goswami, T. and N. C. Andrews (2006). "Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing." J Biol Chem 281(39): 28494-28498. Graham, R. M., A. C. Chua, C. E. Herbison, J. K. Olynyk and D. Trinder (2007). "Liver iron transport." World J Gastroenterol 13(35): 4725-4736. Grootveld, M., J. D. Bell, B. Halliwell, O. I. Aruoma, A. Bomford and P. J. Sadler (1989). "Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy." J Biol Chem 264(8): 4417-4422.
166
Gunshin, H., B. Mackenzie, U. V. Berger, Y. Gunshin, M. F. Romero, W. F. Boron, S. Nussberger, J. L. Gollan and M. A. Hediger (1997). "Cloning and characterization of a mammalian proton-coupled metal-ion transporter." Nature 388(6641): 482-488. Gunshin, H., C. N. Starr, C. Direnzo, M. D. Fleming, J. Jin, E. L. Greer, V. M. Sellers, S. M. Galica and N. C. Andrews (2005). "Cybrd1 (duodenal cytochrome b) is not necessary for dietary iron absorption in mice." Blood 106(8): 2879-2883. Gunshin, H., Y. Fujiwara, A. O. Custodio, C. Direnzo, S. Robine and N. C. Andrews (2005). "Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver." J Clin Invest 115(5): 1258-1266. Guo, J. H., S. H. Juan and S. D. Aust (1998). "Suppression of cell growth by heavy chain ferritin." Biochem Biophys Res Commun 242(1): 39-45. Gutteridge, J. M., D. A. Rowley, E. Griffiths and B. Halliwell (1985). "Low-molecular-weight iron complexes and oxygen radical reactions in idiopathic haemochromatosis." Clin Sci (Lond) 68(4): 463-467. Halliwell, B. and J. M. Gutteridge (1984). "Oxygen toxicity, oxygen radicals, transition metals and disease." Biochem J 219(1): 1-14. Hansen, S. L., N. Trakooljul, H. C. Liu, A. J. Moeser and J. W. Spears (2009). "Iron transporters are differentially regulated by dietary iron, and modifications are associated with changes in manganese metabolism in young pigs." J Nutr 139(8): 1474-1479. Harding, C., Heuser, J., Stahl, P. (1983). "Receptor-mediated endocytosis of transferrin and recylcing of the transferrin receptor in rat reticulocytes." Journal of Cell Biology 97: 329-339. Harrison-Findik, D. D., E. Klein, C. Crist, J. Evans, N. Timchenko and J. Gollan (2007). "Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol." Hepatology 46(6): 1979-1985. Harrison, P. M. and P. Arosio (1996). "The ferritins: molecular properties, iron storage function and cellular regulation." Biochim Biophys Acta 1275(3): 161-203. Harrison, P. M., Ford, G.C., Rice, D.W., Smith, J.M., Treffry, A., White, J.L. (1987). "Structural and functional studies on ferritins." Biochemical Society Transactions 15: 744-748. Harrison, S. A., and Bacon, B.R. (2003). "Hereditary haemochromatosis: update for 2003." Journal of Hepatology 38: S14-S23. Hausmann, A., J. Lee and K. Pantopoulos (2011). "Redox control of iron regulatory protein 2 stability." FEBS Lett 585(4): 687-692. Hentze, M. W., M. U. Muckenthaler, B. Galy and C. Camaschella (2010). "Two to tango: regulation of Mammalian iron metabolism." Cell 142(1): 24-38. Herrmann, T., M. Muckenthaler, F. van der Hoeven, K. Brennan, S. G. Gehrke, N. Hubert, C. Sergi, H. J. Grone, I. Kaiser, I. Gosch, M. Volkmann, H. D. Riedel, M. W. Hentze, A. F. Stewart and W. Stremmel (2004). "Iron overload in adult Hfe-deficient mice independent of
167
changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin." J Mol Med (Berl) 82(1): 39-48. Hershko, C., G. Graham, G. W. Bates and E. A. Rachmilewitz (1978). "Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity." Br J Haematol 40(2): 255-263. Hershko, C., J. Patz and A. Ronson (2007). "The anemia of achylia gastrica revisited." Blood Cells Mol Dis 39(2): 178-183. Hojyo, S., T. Fukada, S. Shimoda, W. Ohashi, B. H. Bin, H. Koseki and T. Hirano (2011). "The zinc transporter SLC39A14/ZIP14 controls G-protein coupled receptor-mediated signaling required for systemic growth." PLoS One 6(3): e18059. Hu, W. L. and E. Regoeczi (1992). "Hepatic heparan sulphate proteoglycan and the recycling of transferrin." Biochem Cell Biol 70(7): 535-538. Huang, F. W., J. L. Pinkus, G. S. Pinkus, M. D. Fleming and N. C. Andrews (2005). "A mouse model of juvenile hemochromatosis." J Clin Invest 115(8): 2187-2191. Huang, H., M. Constante, A. Layoun and M. M. Santos (2009). "Contribution of STAT3 and SMAD4 pathways to the regulation of hepcidin by opposing stimuli." Blood 113(15): 3593-3599. Hubert, N. and M. W. Hentze (2002). "Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function." Proc Natl Acad Sci U S A 99(19): 12345-12350. Huebers, H. A. and C. A. Finch (1987). "The physiology of transferrin and transferrin receptors." Physiol Rev 67(2): 520-582. Hussain, S. P., K. Raja, P. A. Amstad, M. Sawyer, L. J. Trudel, G. N. Wogan, L. J. Hofseth, P. G. Shields, T. R. Billiar, C. Trautwein, T. Hohler, P. R. Galle, D. H. Phillips, R. Markin, A. J. Marrogi and C. C. Harris (2000). "Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases." Proc Natl Acad Sci U S A 97(23): 12770-12775. Hvidberg, V., M. B. Maniecki, C. Jacobsen, P. Hojrup, H. J. Moller and S. K. Moestrup (2005). "Identification of the receptor scavenging hemopexin-heme complexes." Blood 106(7): 2572-2579. Hvidberg, V., C. Jacobsen, R. K. Strong, J. B. Cowland, S. K. Moestrup and N. Borregaard (2005). "The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake." FEBS Lett 579(3): 773-777. Iacopetta, B. J., E. H. Morgan and G. C. Yeoh (1983). "Receptor-mediated endocytosis of transferrin by developing erythroid cells from the fetal rat liver." J Histochem Cytochem 31(2): 336-344. Iancu, T. C. (1992). "Ferritin and hemosiderin in pathological tissues." Electron Microsc Rev 5(2): 209-229.
168
Iancu, T. C. and H. Shiloh (1994). "Morphologic observations in iron overload: an update." Adv Exp Med Biol 356: 255-265. Iancu, T. C., Y. Deugnier, J. W. Halliday, L. W. Powell and P. Brissot (1997). "Ultrastructural sequences during liver iron overload in genetic hemochromatosis." J Hepatol 27(4): 628-638. Inman, R. S., M. M. Coughlan and M. Wessling-Resnick (1994). "Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport." Biochemistry 33(39): 11850-11857. Johnson, M. B. and C. A. Enns (2004). "Diferric transferrin regulates transferrin receptor 2 protein stability." Blood 104(13): 4287-4293. Jomova, K. and M. Valko (2011). "Advances in metal-induced oxidative stress and human disease." Toxicology 283(2-3): 65-87. Jordan, I. and J. Kaplan (1994). "The mammalian transferrin-independent iron transport system may involve a surface ferrireductase activity." Biochem J 302 ( Pt 3): 875-879. Jouihan, H. A., P. A. Cobine, R. C. Cooksey, E. A. Hoagland, S. Boudina, E. D. Abel, D. R. Winge and D. A. McClain (2008). "Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis." Mol Med 14(3-4): 98-108. Kakhlon, O. and Z. I. Cabantchik (2002). "The labile iron pool: characterization, measurement, and participation in cellular processes(1)." Free Radic Biol Med 33(8): 1037-1046. Kautz, L., D. Meynard, A. Monnier, V. Darnaud, R. Bouvet, R. H. Wang, C. Deng, S. Vaulont, J. Mosser, H. Coppin and M. P. Roth (2008). "Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver." Blood 112(4): 1503-1509. Kawabata, H., R. Yang, T. Hirama, P. T. Vuong, S. Kawano, A. F. Gombart and H. P. Koeffler (1999). "Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family." J Biol Chem 274(30): 20826-20832. Kawabata, H., R. S. Germain, P. T. Vuong, T. Nakamaki, J. W. Said and H. P. Koeffler (2000). "Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo." J Biol Chem 275(22): 16618-16625. Kawabata, H., R. S. Germain, T. Ikezoe, X. Tong, E. M. Green, A. F. Gombart and H. P. Koeffler (2001). "Regulation of expression of murine transferrin receptor 2." Blood 98(6): 1949-1954. Kawabata, H., R. E. Fleming, D. Gui, S. Y. Moon, T. Saitoh, J. O'Kelly, Y. Umehara, Y. Wano, J. W. Said and H. P. Koeffler (2005). "Expression of hepcidin is down-regulated in TfR2 mutant mice manifesting a phenotype of hereditary hemochromatosis." Blood 105(1): 376-381. Keel, S. B., R. T. Doty, Z. Yang, J. G. Quigley, J. Chen, S. Knoblaugh, P. D. Kingsley, I. De Domenico, M. B. Vaughn, J. Kaplan, J. Palis and J. L. Abkowitz (2008). "A heme export
169
protein is required for red blood cell differentiation and iron homeostasis." Science 319(5864): 825-828. Kim, G., P. Khanal, S. C. Lim, H. J. Yun, S. G. Ahn, S. H. Ki and H. S. Choi (2012). "Interleukin-17 induces AP-1 activity and cellular transformation via upregulation of tumor progression locus 2 activity." Carcinogenesis 34(2): 341-350. Kingsley, D. M. (1994). "The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms." Genes Dev 8(2): 133-146. Klausner, R. D., G. Ashwell, J. van Renswoude, J. B. Harford and K. R. Bridges (1983). "Binding of apotransferrin to K562 cells: explanation of the transferrin cycle." Proc Natl Acad Sci U S A 80(8): 2263-2266. Knutson, M. D., M. R. Vafa, D. J. Haile and M. Wessling-Resnick (2003). "Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages." Blood 102(12): 4191-4197. Kondo, H., K. Saito, J. P. Grasso and P. Aisen (1988). "Iron metabolism in the erythrophagocytosing Kupffer cell." Hepatology 8(1): 32-38. Konijn, A. M., Hershko, C. (1977). "Ferritin synthesis in inflammation. Pathogenesis of impaired iron release." British Journal of Haematology 37: 7-16. Koren, A., D. Fink, O. Admoni, Y. Tennenbaum-Rakover and C. Levin (2010). "Non-transferrin-bound labile plasma iron and iron overload in sickle-cell disease: a comparative study between sickle-cell disease and beta-thalassemic patients." Eur J Haematol 84(1): 72-78. Kozyraki, R., J. Fyfe, P. J. Verroust, C. Jacobsen, A. Dautry-Varsat, J. Gburek, T. E. Willnow, E. I. Christensen and S. K. Moestrup (2001). "Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia." Proc Natl Acad Sci U S A 98(22): 12491-12496. Krause, A., S. Neitz, H. J. Magert, A. Schulz, W. G. Forssmann, P. Schulz-Knappe and K. Adermann (2000). "LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity." FEBS Lett 480(2-3): 147-150. Krijt, J., M. Vokurka, K. T. Chang and E. Necas (2004). "Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin." Blood 104(13): 4308-4310. Kristiansen, M., J. H. Graversen, C. Jacobsen, O. Sonne, H. J. Hoffman, S. K. Law and S. K. Moestrup (2001). "Identification of the haemoglobin scavenger receptor." Nature 409(6817): 198-201. Kruszewski, M. (2003). "Labile iron pool: the main determinant of cellular response to oxidative stress." Mutat Res 531(1-2): 81-92. Lakshmi Devi, S. and C. V. Anuradha (2010). "Mitochondrial damage, cytotoxicity and apoptosis in iron-potentiated alcoholic liver fibrosis: amelioration by taurine." Amino Acids 38(3): 869-879.
170
Lambert, L. A. (2012). "Molecular evolution of the transferrin family and associated receptors." Biochim Biophys Acta 1820(3): 244-255. Lee, D. H., D. Y. Liu, D. R. Jacobs, Jr., H. R. Shin, K. Song, I. K. Lee, B. Kim and R. C. Hider (2006). "Common presence of non-transferrin-bound iron among patients with type 2 diabetes." Diabetes Care 29(5): 1090-1095. Lee, P. L., Gelbart, T., West, C., Halloran, C., Beutler, E. (1998). "The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms." Blood Cells, Molecules, and Diseases 24: 199-215. Lesbordes-Brion, J. C., L. Viatte, M. Bennoun, D. Q. Lou, G. Ramey, C. Houbron, G. Hamard, A. Kahn and S. Vaulont (2006). "Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis." Blood 108(4): 1402-1405. Levy, J. E., L. K. Montross, D. E. Cohen, M. D. Fleming and N. C. Andrews (1999). "The C282Y mutation causing hereditary hemochromatosis does not produce a null allele." Blood 94(1): 9-11. Li, J. Y., N. Paragas, R. M. Ned, A. Qiu, M. Viltard, T. Leete, I. R. Drexler, X. Chen, S. Sanna-Cherchi, F. Mohammed, D. Williams, C. S. Lin, K. M. Schmidt-Ott, N. C. Andrews and J. Barasch (2009). "Scara5 is a ferritin receptor mediating non-transferrin iron delivery." Dev Cell 16(1): 35-46. Li, L., C. J. Fang, J. C. Ryan, E. C. Niemi, J. A. Lebron, P. J. Bjorkman, H. Arase, F. M. Torti, S. V. Torti, M. C. Nakamura and W. E. Seaman (2009). "Binding and uptake of H-ferritin are mediated by human transferrin receptor-1." Proc Natl Acad Sci U S A 107(8): 3505-3510. Lichten, L. A. and R. J. Cousins (2009). "Mammalian zinc transporters: nutritional and physiologic regulation." Annu Rev Nutr 29: 153-176. Lichten, L. A., J. P. Liuzzi and R. J. Cousins (2009). "Interleukin-1beta contributes via nitric oxide to the upregulation and functional activity of the zinc transporter Zip14 (Slc39a14) in murine hepatocytes." Am J Physiol Gastrointest Liver Physiol 296(4): G860-867. Lin, E. and P. C. Adams (1991). "Biochemical liver profile in hemochromatosis. A survey of 100 patients." J Clin Gastroenterol 13(3): 316-320. Lin, M., R. A. Rippe, O. Niemela, G. Brittenham and H. Tsukamoto (1997). "Role of iron in NF-kappa B activation and cytokine gene expression by rat hepatic macrophages." Am J Physiol 272(6 Pt 1): G1355-1364. Lipscombe, D., T. D. Helton and W. Xu (2004). "L-type calcium channels: the low down." J Neurophysiol 92(5): 2633-2641. Liu, X. B., N. B. Nguyen, K. D. Marquess, F. Yang and D. J. Haile (2005). "Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages." Blood Cells Mol Dis 35(1): 47-56. Liuzzi, J. P., L. A. Lichten, S. Rivera, R. K. Blanchard, T. B. Aydemir, M. D. Knutson, T. Ganz and R. J. Cousins (2005). "Interleukin-6 regulates the zinc transporter Zip14 in liver
171
and contributes to the hypozincemia of the acute-phase response." Proc Natl Acad Sci U S A 102(19): 6843-6848. Liuzzi, J. P., F. Aydemir, H. Nam, M. D. Knutson and R. J. Cousins (2006). "Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells." Proc Natl Acad Sci U S A 103(37): 13612-13617. Livrea, M. A., L. Tesoriere, A. M. Pintaudi, A. Calabrese, A. Maggio, H. J. Freisleben, D. D'Arpa, R. D'Anna and A. Bongiorno (1996). "Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants." Blood 88(9): 3608-3614. Mackinnon, M., C. Clayton, J. Plummer, M. Ahern, P. Cmielewski, A. Ilsley and P. Hall (1995). "Iron overload facilitates hepatic fibrosis in the rat alcohol/low-dose carbon tetrachloride model." Hepatology 21(4): 1083-1088. Malicka-Blaszkiewicz, M. and A. Kubicz (1979). "Partial purification and some properties of a liver alkaline ribonuclease from the frog Rana esculenta." Acta Biochim Pol 26(3): 275-283. Mao, X., B. E. Kim, F. Wang, D. J. Eide and M. J. Petris (2007). "A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity." J Biol Chem 282(10): 6992-7000. Mastrogiannaki, M., P. Matak, B. Keith, M. C. Simon, S. Vaulont and C. Peyssonnaux (2009). "HIF-2alpha, but not HIF-1alpha, promotes iron absorption in mice." J Clin Invest 119(5): 1159-1166. Matak, P., T. B. Chaston, B. Chung, S. K. Srai, A. T. McKie and P. A. Sharp (2009). "Activated macrophages induce hepcidin expression in HuH7 hepatoma cells." Haematologica 94(6): 773-780. Matayatsuk, C., C. Y. Lee, R. W. Kalpravidh, P. Sirankapracha, P. Wilairat, S. Fucharoen and B. Halliwell (2007). "Elevated F2-isoprostanes in thalassemic patients." Free Radic Biol Med 43(12): 1649-1655. Matsuzaki, K. (2012). "Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells." Cell Tissue Res 347(1): 225-243. May, J. M., Z. C. Qu and S. Mendiratta (1999). "Role of ascorbic acid in transferrin-independent reduction and uptake of iron by U-937 cells." Biochem Pharmacol 57(11): 1275-1282. May, P., Linder, P., and D. Williams (1977). "Computer simulation of metal-ion equilibria in biofluids: models for the low-molecular-weight complex distribution of calcium(II), magnesium(II), manganese(II), iron(III), copper(II), zinc(II), and lead(II) ions in human blood plasma." Journal of the Chemical Society, Dalton Transactions(6): 588-595. McCord, J. M. and I. Fridovich (1988). "Superoxide dismutase: the first twenty years (1968-1988)." Free Radic Biol Med 5(5-6): 363-369.
172
McKie, A. T., P. Marciani, A. Rolfs, K. Brennan, K. Wehr, D. Barrow, S. Miret, A. Bomford, T. J. Peters, F. Farzaneh, M. A. Hediger, M. W. Hentze and R. J. Simpson (2000). "A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation." Mol Cell 5(2): 299-309. McKie, A. T., D. Barrow, G. O. Latunde-Dada, A. Rolfs, G. Sager, E. Mudaly, M. Mudaly, C. Richardson, D. Barlow, A. Bomford, T. J. Peters, K. B. Raja, S. Shirali, M. A. Hediger, F. Farzaneh and R. J. Simpson (2001). "An iron-regulated ferric reductase associated with the absorption of dietary iron." Science 291(5509): 1755-1759. McLachlan, S., S. M. Lee, T. M. Steele, P. L. Hawthorne, M. A. Zapala, E. Eskin, N. J. Schork, G. J. Anderson and C. D. Vulpe (2011). "In silico QTL mapping of basal liver iron levels in inbred mouse strains." Physiol Genomics 43(3): 136-147. McLaren, C. E., J. C. Barton, V. R. Gordeuk, L. Wu, P. C. Adams, D. M. Reboussin, M. Speechley, H. Chang, R. T. Acton, E. L. Harris, A. M. Ruggiero and O. Castro (2007). "Determinants and characteristics of mean corpuscular volume and hemoglobin concentration in white HFE C282Y homozygotes in the hemochromatosis and iron overload screening study." Am J Hematol 82(10): 898-905. Meynard, D., V. Vaja, C. C. Sun, E. Corradini, S. Chen, C. Lopez-Otin, L. Grgurevic, C. C. Hong, M. Stirnberg, M. Gutschow, S. Vukicevic, J. L. Babitt and H. Y. Lin (2011). "Regulation of TMPRSS6 by BMP6 and iron in human cells and mice." Blood 118(3): 747-756. Miossec, P., T. Korn and V. K. Kuchroo (2009). "Interleukin-17 and type 17 helper T cells." N Engl J Med 361(9): 888-898. Miskimins, W. K., A. McClelland, M. P. Roberts and F. H. Ruddle (1986). "Cell proliferation and expression of the transferrin receptor gene: promoter sequence homologies and protein interactions." J Cell Biol 103(5): 1781-1788. Miyazaki, E., J. Kato, M. Kobune, K. Okumura, K. Sasaki, N. Shintani, P. Arosio and Y. Niitsu (2002). "Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload." Gut 50(3): 413-419. Mizukami, S., R. Ichimura, S. Kemmochi, L. Wang, E. Taniai, K. Mitsumori and M. Shibutani (2010). "Tumor promotion by copper-overloading and its enhancement by excess iron accumulation involving oxidative stress responses in the early stage of a rat two-stage hepatocarcinogenesis model." Chem Biol Interact 185(3): 189-201. Mleczko-Sanecka, K., G. Casanovas, A. Ragab, K. Breitkopf, A. Muller, M. Boutros, S. Dooley, M. W. Hentze and M. U. Muckenthaler (2010). "SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression." Blood 115(13): 2657-2665. Morgan, E. H. (1981). "Transferrin, biochemistry, physiology and clinical significance." Molecular Aspects of Medicine 4(1): 1-123. Morgan, E. H. and E. Baker (1986). "Iron uptake and metabolism by hepatocytes." Fed Proc 45(12): 2810-2816. Morgan, E. H. (1996). Iron metabolism and transport. Hepatology: A Textbook of Liver Disease. Z. D. a. B. TD. Philadelphia, Saunders Company.
173
Mori, K., H. T. Lee, D. Rapoport, I. R. Drexler, K. Foster, J. Yang, K. M. Schmidt-Ott, X. Chen, J. Y. Li, S. Weiss, J. Mishra, F. H. Cheema, G. Markowitz, T. Suganami, K. Sawai, M. Mukoyama, C. Kunis, V. D'Agati, P. Devarajan and J. Barasch (2005). "Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury." J Clin Invest 115(3): 610-621. Mori, T. A., K. D. Croft, I. B. Puddey and L. J. Beilin (1999). "An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry." Anal Biochem 268(1): 117-125. Morikawa, K., F. Oseko and S. Morikawa (1995). "A role for ferritin in hematopoiesis and the immune system." Leuk Lymphoma 18(5-6): 429-433. Moroishi, T., M. Nishiyama, Y. Takeda, K. Iwai and K. I. Nakayama (2011). "The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo." Cell Metab 14(3): 339-351. Muckenthaler, M. U., B. Galy and M. W. Hentze (2008). "Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network." Annu Rev Nutr 28: 197-213. Murray, M. J., A. B. Murray, M. B. Murray and C. J. Murray (1978). "The adverse effect of iron repletion on the course of certain infections." Br Med J 2(6145): 1113-1115. Nair, J., A. Barbin, Y. Guichard and H. Bartsch (1995). "1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabeling." Carcinogenesis 16(3): 613-617. Nair, J., P. L. Carmichael, R. C. Fernando, D. H. Phillips, A. J. Strain and H. Bartsch (1998). "Lipid peroxidation-induced etheno-DNA adducts in the liver of patients with the genetic metal storage disorders Wilson's disease and primary hemochromatosis." Cancer Epidemiol Biomarkers Prev 7(5): 435-440. Nairz, M., I. Theurl, A. Schroll, M. Theurl, G. Fritsche, E. Lindner, M. Seifert, M. L. Crouch, K. Hantke, S. Akira, F. C. Fang and G. Weiss (2009). "Absence of functional Hfe protects mice from invasive Salmonella enterica serovar Typhimurium infection via induction of lipocalin-2." Blood 114(17): 3642-3651. Nairz, M., A. Schroll, A. R. Moschen, T. Sonnweber, M. Theurl, I. Theurl, N. Taub, C. Jamnig, D. Neurauter, L. A. Huber, H. Tilg, P. L. Moser and G. Weiss (2011). "Erythropoietin contrastingly affects bacterial infection and experimental colitis by inhibiting nuclear factor-kappaB-inducible immune pathways." Immunity 34(1): 61-74. Nam, H., C. Y. Wang, L. Zhang, W. Zhang, S. Hojyo, T. Fukada and M. Knutson (2013). "ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders." Haematologica. Nemeth, E., M. S. Tuttle, J. Powelson, M. B. Vaughn, A. Donovan, D. M. Ward, T. Ganz and J. Kaplan (2004). "Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization." Science 306(5704): 2090-2093.
174
Nemeth, E., S. Rivera, V. Gabayan, C. Keller, S. Taudorf, B. K. Pedersen and T. Ganz (2004). "IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin." J Clin Invest 113(9): 1271-1276. Nemeth, E. and T. Ganz (2006). "Hepcidin and iron-loading anemias." Haematologica 91(6): 727-732. Nicolas, G., M. Bennoun, I. Devaux, C. Beaumont, B. Grandchamp, A. Kahn and S. Vaulont (2001). "Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice." Proc Natl Acad Sci U S A 98(15): 8780-8785. Nicolas, G., M. Bennoun, A. Porteu, S. Mativet, C. Beaumont, B. Grandchamp, M. Sirito, M. Sawadogo, A. Kahn and S. Vaulont (2002). "Severe iron deficiency anemia in transgenic mice expressing liver hepcidin." Proc Natl Acad Sci U S A 99(7): 4596-4601. Nicolas, G., C. Chauvet, L. Viatte, J. L. Danan, X. Bigard, I. Devaux, C. Beaumont, A. Kahn and S. Vaulont (2002). "The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation." J Clin Invest 110(7): 1037-1044. Niederkofler, V., R. Salie and S. Arber (2005). "Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload." J Clin Invest 115(8): 2180-2186. Nili, M., U. Shinde and P. Rotwein (2010). "Soluble repulsive guidance molecule c/hemojuvelin is a broad spectrum bone morphogenetic protein (BMP) antagonist and inhibits both BMP2- and BMP6-mediated signaling and gene expression." J Biol Chem 285(32): 24783-24792. Nilsson, R., I. J. Schultz, E. L. Pierce, K. A. Soltis, A. Naranuntarat, D. M. Ward, J. M. Baughman, P. N. Paradkar, P. D. Kingsley, V. C. Culotta, J. Kaplan, J. Palis, B. H. Paw and V. K. Mootha (2009). "Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis." Cell Metab 10(2): 119-130. Noetzli, L. J., J. Papudesi, T. D. Coates and J. C. Wood (2009). "Pancreatic iron loading predicts cardiac iron loading in thalassemia major." Blood 114(19): 4021-4026. O'Connell, M. J., R. J. Ward, H. Baum and T. J. Peters (1985). "The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes." Biochem J 229(1): 135-139. Ohgami, R. S., D. R. Campagna, E. L. Greer, B. Antiochos, A. McDonald, J. Chen, J. J. Sharp, Y. Fujiwara, J. E. Barker and M. D. Fleming (2005). "Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells." Nat Genet 37(11): 1264-1269. Oliveira, S. J., J. P. Pinto, G. Picarote, V. M. Costa, F. Carvalho, M. Rangel, M. de Sousa and S. F. de Almeida (2009). "ER stress-inducible factor CHOP affects the expression of hepcidin by modulating C/EBPalpha activity." PLoS One 4(8): e6618. Olynyk, J. K., T. G. St Pierre, R. S. Britton, E. M. Brunt and B. R. Bacon (2005). "Duration of hepatic iron exposure increases the risk of significant fibrosis in hereditary hemochromatosis: a new role for magnetic resonance imaging." Am J Gastroenterol 100(4): 837-841.
175
Olynyk, J. K., D. Trinder, G. A. Ramm, R. S. Britton and B. R. Bacon (2008). "Hereditary hemochromatosis in the post-HFE era." Hepatology 48(3): 991-1001. Olynyk, J. K., Cullen, D.J., Aquilia, S., Rossi, E., Summerville, L., Powell, L.W. (1999). "A population based study of the clinical expression of haemochromatosis gene." The New England Journal of Medicine 341: 718-724. Oudit, G. Y., H. Sun, M. G. Trivieri, S. E. Koch, F. Dawood, C. Ackerley, M. Yazdanpanah, G. J. Wilson, A. Schwartz, P. P. Liu and P. H. Backx (2003). "L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy." Nat Med 9(9): 1187-1194. Ouyang, Q., M. Bommakanti and W. K. Miskimins (1993). "A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways." Mol Cell Biol 13(3): 1796-1804. Pak, M., M. A. Lopez, V. Gabayan, T. Ganz and S. Rivera (2006). "Suppression of hepcidin during anemia requires erythropoietic activity." Blood 108(12): 3730-3735. Paradkar, P. N., I. De Domenico, N. Durchfort, I. Zohn, J. Kaplan and D. M. Ward (2008). "Iron depletion limits intracellular bacterial growth in macrophages." Blood 112(3): 866-874. Park, C. H., E. V. Valore, A. J. Waring and T. Ganz (2001). "Hepcidin, a urinary antimicrobial peptide synthesized in the liver." J Biol Chem 276(11): 7806-7810. Parkkila, S., A. Waheed, R. S. Britton, B. R. Bacon, X. Y. Zhou, S. Tomatsu, R. E. Fleming and W. S. Sly (1997). "Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis." Proc Natl Acad Sci U S A 94(24): 13198-13202. Parola, M. and G. Robino (2001). "Oxidative stress-related molecules and liver fibrosis." J Hepatol 35(2): 297-306. Peslova, G., J. Petrak, K. Kuzelova, I. Hrdy, P. Halada, P. W. Kuchel, S. Soe-Lin, P. Ponka, R. Sutak, E. Becker, M. L. Huang, Y. Suryo Rahmanto, D. R. Richardson and D. Vyoral (2009). "Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood." Blood 113(24): 6225-6236. Philpott, C. C. (2002). "Molecular aspects of iron absorption: insights into the role of HFE in haemochromatosis." Hepatology 35: 993-1001. Picard, V., G. Govoni, N. Jabado and P. Gros (2000). "Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool." J Biol Chem 275(46): 35738-35745. Pietrangelo, A., E. Rocchi, L. Schiaffonati, E. Ventura and G. Cairo (1990). "Liver gene expression during chronic dietary iron overload in rats." Hepatology 11(5): 798-804. Pietrangelo, A. (2004). "The ferroportin disease." Blood Cells, Molecules & Diseases 32: 131-138. Pietrangelo, A. (2004). "The ferroportin disease." Blood Cells Mol Dis 32(1): 131-138.
176
Pietrangelo, A. (2004). "Hereditary hemochromatosis--a new look at an old disease." N Engl J Med 350(23): 2383-2397. Pietrangelo, A., A. Caleffi, J. Henrion, F. Ferrara, E. Corradini, H. Kulaksiz, W. Stremmel, P. Andreone and C. Garuti (2005). "Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes." Gastroenterology 128(2): 470-479. Pietrangelo, A. (2006). "Hereditary hemochromatosis." Biochim Biophys Acta 1763(7): 700-710. Pietrangelo, A., U. Dierssen, L. Valli, C. Garuti, A. Rump, E. Corradini, M. Ernst, C. Klein and C. Trautwein (2007). "STAT3 is required for IL-6-gp130-dependent activation of hepcidin in vivo." Gastroenterology 132(1): 294-300. Pigeon, C., G. Ilyin, B. Courselaud, P. Leroyer, B. Turlin, P. Brissot and O. Loreal (2001). "A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload." J Biol Chem 276(11): 7811-7819. Pinilla-Tenas, J. J., B. K. Sparkman, A. Shawki, A. C. Illing, C. J. Mitchell, N. Zhao, J. P. Liuzzi, R. J. Cousins, M. D. Knutson and B. Mackenzie (2011). "Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron." Am J Physiol Cell Physiol 301(4): C862-871. Pinto, J. P., S. Ribeiro, H. Pontes, S. Thowfeequ, D. Tosh, F. Carvalho and G. Porto (2008). "Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha." Blood 111(12): 5727-5733. Piperno, A. (1998). "Classification and diagnosis of iron overload." Haematologica 83(5): 447-455. Ponka, P. (1997). "Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells." Blood 89(1): 1-25. Ponka, P., C. Beaumont and D. R. Richardson (1998). "Function and regulation of transferrin and ferritin." Semin Hematol 35(1): 35-54. Pootrakul, P., W. Breuer, M. Sametband, P. Sirankapracha, C. Hershko and Z. I. Cabantchik (2004). "Labile plasma iron (LPI) as an indicator of chelatable plasma redox activity in iron-overloaded beta-thalassemia/HbE patients treated with an oral chelator." Blood 104(5): 1504-1510. Porto, G., H. Alves, P. Rodrigues, J. M. Cabeda, C. Portal, A. Ruivo, B. Justica, R. Wolff and M. De Sousa (1998). "Major histocompatibility complex class I associations in iron overload: evidence for a new link between the HFE H63D mutation, HLA-A29, and non-classical forms of hemochromatosis." Immunogenetics 47(5): 404-410. Pozo, A. L., E. M. Godfrey and K. M. Bowles (2009). "Splenomegaly: investigation, diagnosis and management." Blood Rev 23(3): 105-111. Prakash, M., S. Upadhya and R. Prabhu (2005). "Serum non-transferrin bound iron in hemodialysis patients not receiving intravenous iron." Clin Chim Acta 360(1-2): 194-198.
177
Qian, Z. M. and E. H. Morgan (1992). "Changes in the uptake of transferrin-free and transferrin-bound iron during reticulocyte maturation in vivo and in vitro." Biochim Biophys Acta 1135(1): 35-43. Qiu, A., M. Jansen, A. Sakaris, S. H. Min, S. Chattopadhyay, E. Tsai, C. Sandoval, R. Zhao, M. H. Akabas and I. D. Goldman (2006). "Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption." Cell 127(5): 917-928. Raffin, S. B., Woo, C.H., Roost, K.T., Price, D.C., Schmid, R. (1974). "Intestinal absorption of hemoglobin iron-heme cleavage by mucosal heme oxygenase." Journal of Clinical Investigation 54: 1344-1352. Raje, C. I., S. Kumar, A. Harle, J. S. Nanda and M. Raje (2007). "The macrophage cell surface glyceraldehyde-3-phosphate dehydrogenase is a novel transferrin receptor." J Biol Chem 282(5): 3252-3261. Ramey, G., J. C. Deschemin and S. Vaulont (2009). "Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes." Haematologica 94(6): 765-772. Ramirez, J. M., O. Schaad, S. Durual, D. Cossali, M. Docquier, P. Beris, P. Descombes and T. Matthes (2009). "Growth differentiation factor 15 production is necessary for normal erythroid differentiation and is increased in refractory anaemia with ring-sideroblasts." Br J Haematol 144(2): 251-262. Ramm, G. A., D. H. Crawford, L. W. Powell, N. I. Walker, L. M. Fletcher and J. W. Halliday (1997). "Hepatic stellate cell activation in genetic haemochromatosis. Lobular distribution, effect of increasing hepatic iron and response to phlebotomy." J Hepatol 26(3): 584-592. Ramm, G. A. and R. G. Ruddell (2010). "Iron homeostasis, hepatocellular injury, and fibrogenesis in hemochromatosis: the role of inflammation in a noninflammatory liver disease." Semin Liver Dis 30(3): 271-287. Ramos, P., E. Guy, N. Chen, C. C. Proenca, S. Gardenghi, C. Casu, A. Follenzi, N. Van Rooijen, R. W. Grady, M. de Sousa and S. Rivella (2011). "Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis." Blood 117(4): 1379-1389. Randell, E. W., J. G. Parkes, N. F. Olivieri and D. M. Templeton (1994). "Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron." J Biol Chem 269(23): 16046-16053. Reardon, T. F. and D. G. Allen (2009). "Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance." Exp Physiol 94(6): 720-730. Richardson, D. R., D. J. Lane, E. M. Becker, M. L. Huang, M. Whitnall, Y. Suryo Rahmanto, A. D. Sheftel and P. Ponka (2010). "Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol." Proc Natl Acad Sci U S A 107(24): 10775-10782.
178
Robach, P., S. Recalcati, D. Girelli, C. Gelfi, N. J. Aachmann-Andersen, J. J. Thomsen, A. M. Norgaard, A. Alberghini, N. Campostrini, A. Castagna, A. Vigano, P. Santambrogio, T. Kempf, K. C. Wollert, S. Moutereau, C. Lundby and G. Cairo (2009). "Alterations of systemic and muscle iron metabolism in human subjects treated with low-dose recombinant erythropoietin." Blood 113(26): 6707-6715. Robb, A. and M. Wessling-Resnick (2004). "Regulation of transferrin receptor 2 protein levels by transferrin." Blood 104(13): 4294-4299. Roetto, A., G. Papanikolaou, M. Politou, F. Alberti, D. Girelli, J. Christakis, D. Loukopoulos and C. Camaschella (2003). "Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis." Nat Genet 33(1): 21-22. Rouault, T. A. (2006). "The role of iron regulatory proteins in mammalian iron homeostasis and disease." Nat Chem Biol 2(8): 406-414. Rueda Adel, C., N. C. Grande, E. A. Fernandez, R. Enriquez de Salamanca, L. A. Sala and M. J. Jimenez (2011). "Mutations in HFE and TFR2 genes in a Spanish patient with hemochromatosis." Rev Esp Enferm Dig 103(7): 379-382. Ryter, S. W. and R. M. Tyrrell (2000). "The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties." Free Radic Biol Med 28(2): 289-309. Sahlstedt, L., L. von Bonsdorff, F. Ebeling, J. Parkkinen, E. Juvonen and T. Ruutu (2009). "Non-transferrin-bound iron in haematological patients during chemotherapy and conditioning for autologous stem cell transplantation." Eur J Haematol 83(5): 455-459. Salama, M. F., H. K. Bayele and S. S. Srai (2012). "Tumour necrosis factor alpha downregulates human hemojuvelin expression via a novel response element within its promoter." J Biomed Sci 19: 83. Schmidt-Ott, K. M., K. Mori, A. Kalandadze, J. Y. Li, N. Paragas, T. Nicholas, P. Devarajan and J. Barasch (2006). "Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia." Curr Opin Nephrol Hypertens 15(4): 442-449. Seiser, C., S. Teixeira and L. C. Kuhn (1993). "Interleukin-2-dependent transcriptional and post-transcriptional regulation of transferrin receptor mRNA." J Biol Chem 268(18): 13074-13080. Selden, C., M. Owen, J. M. Hopkins and T. J. Peters (1980). "Studies on the concentration and intracellular localization of iron proteins in liver biopsy specimens from patients with iron overload with special reference to their role in lysosomal disruption." Br J Haematol 44(4): 593-603. Severance, S. and I. Hamza (2009). "Trafficking of heme and porphyrins in metazoa." Chem Rev 109(10): 4596-4616. Shah, Y. M., T. Matsubara, S. Ito, S. H. Yim and F. J. Gonzalez (2009). "Intestinal hypoxia-inducible transcription factors are essential for iron absorption following iron deficiency." Cell Metab 9(2): 152-164.
179
Shanmugam, N. K., S. Ellenbogen, E. Trebicka, L. Wang, S. Mukhopadhyay, A. Lacy-Hulbert, C. A. Gallini, W. S. Garrett and B. J. Cherayil (2012). "Tumor necrosis factor alpha inhibits expression of the iron regulating hormone hepcidin in murine models of innate colitis." PLoS One 7(5): e38136. Shaw, G. C., J. J. Cope, L. Li, K. Corson, C. Hersey, G. E. Ackermann, B. Gwynn, A. J. Lambert, R. A. Wingert, D. Traver, N. S. Trede, B. A. Barut, Y. Zhou, E. Minet, A. Donovan, A. Brownlie, R. Balzan, M. J. Weiss, L. L. Peters, J. Kaplan, L. I. Zon and B. H. Paw (2006). "Mitoferrin is essential for erythroid iron assimilation." Nature 440(7080): 96-100. Shayeghi, M., G. O. Latunde-Dada, J. S. Oakhill, A. H. Laftah, K. Takeuchi, N. Halliday, Y. Khan, A. Warley, F. E. McCann, R. C. Hider, D. M. Frazer, G. J. Anderson, C. D. Vulpe, R. J. Simpson and A. T. McKie (2005). "Identification of an intestinal heme transporter." Cell 122(5): 789-801. Sheth, S., and Brittenham, G.M. (2000). "Genetic disorders affecting proteins or iron metabolism: clinical implications." Annual Review of Medicine 51: 443-464. Shindo, M., Y. Torimoto, H. Saito, W. Motomura, K. Ikuta, K. Sato, Y. Fujimoto and Y. Kohgo (2006). "Functional role of DMT1 in transferrin-independent iron uptake by human hepatocyte and hepatocellular carcinoma cell, HLF." Hepatol Res 35(3): 152-162. Siddique, A. and K. V. Kowdley (2012). "Review article: the iron overload syndromes." Aliment Pharmacol Ther 35(8): 876-893. Silva, A. M. and R. C. Hider (2009). "Influence of non-enzymatic post-translation modifications on the ability of human serum albumin to bind iron. Implications for non-transferrin-bound iron speciation." Biochim Biophys Acta 1794(10): 1449-1458. Silvestri, L., A. Pagani, A. Nai, I. De Domenico, J. Kaplan and C. Camaschella (2008). "The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin." Cell Metab 8(6): 502-511. Smith, C. P. and F. Thevenod (2009). "Iron transport and the kidney." Biochim Biophys Acta 1790(7): 724-730. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk (1985). "Measurement of protein using bicinchoninic acid." Anal Biochem 150(1): 76-85. Sochaski, M. A., W. J. Bartfay, S. R. Thorpe, J. W. Baynes, E. Bartfay, D. C. Lehotay and P. P. Liu (2002). "Lipid peroxidation and protein modification in a mouse model of chronic iron overload." Metabolism 51(5): 645-651. Spivak, J. L. (2000). "The blood in systemic disorders." Lancet 355(9216): 1707-1712. Sposi, N. M., L. Cianetti, E. Tritarelli, E. Pelosi, S. Militi, T. Barberi, M. Gabbianelli, E. Saulle, L. Kuhn, C. Peschle and U. Testa (2000). "Mechanisms of differential transferrin receptor expression in normal hematopoiesis." Eur J Biochem 267(23): 6762-6774.
180
Srivastava, G., I. A. Borthwick, D. J. Maguire, C. J. Elferink, M. J. Bawden, J. F. Mercer and B. K. May (1988). "Regulation of 5-aminolevulinate synthase mRNA in different rat tissues." J Biol Chem 263(11): 5202-5209. Stal, P., U. Broome, A. Scheynius, R. Befrits and R. Hultcrantz (1995). "Kupffer cell iron overload induces intercellular adhesion molecule-1 expression on hepatocytes in genetic hemochromatosis." Hepatology 21(5): 1308-1316. Steinbicker, A. U., T. B. Bartnikas, L. K. Lohmeyer, P. Leyton, C. Mayeur, S. M. Kao, A. E. Pappas, R. T. Peterson, D. B. Bloch, P. B. Yu, M. D. Fleming and K. D. Bloch (2011). "Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice." Blood 118(15): 4224-4230. Su, D., J. M. May, M. J. Koury and H. Asard (2006). "Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling." J Biol Chem 281(52): 39852-39859. Su, M. A., Trenor, C.C., Fleming, J.C., Fleming, M.D. and Andrews, N.C. (1998). "The G185R mutation disrupts function of the iron transporter Nramp2." Blood 92: 2157-2163. Subramaniam, V. N., C. J. McDonald, L. Ostini, P. E. Lusby, L. F. Wockner, G. A. Ramm and D. F. Wallace (2012). "Hepatic iron deposition does not predict extrahepatic iron loading in mouse models of hereditary hemochromatosis." Am J Pathol 181(4): 1173-1179. Sverko, V., T. Balog, S. Sobocanec, M. Gavella and T. Marotti (2002). "Age-associated alteration of lipid peroxidation and superoxide dismutase activity in CBA and AKR mice." Exp Gerontol 37(8-9): 1031-1039. Tacchini, L., L. Bianchi, A. Bernelli-Zazzera and G. Cairo (1999). "Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation." J Biol Chem 274(34): 24142-24146. Takagi, H., D. Shi, Y. Ha, N. M. Allewell and E. C. Theil (1998). "Localized unfolding at the junction of three ferritin subunits. A mechanism for iron release?" J Biol Chem 273(30): 18685-18688. Tan, T. C., D. H. Crawford, L. A. Jaskowski, T. M. Murphy, M. L. Heritage, V. N. Subramaniam, A. D. Clouston, G. J. Anderson and L. M. Fletcher (2011). "Altered lipid metabolism in Hfe-knockout mice promotes severe NAFLD and early fibrosis." Am J Physiol Gastrointest Liver Physiol 301(5): G865-876. Tanaka, T., C. N. Roy, W. Yao, A. Matteini, R. D. Semba, D. Arking, J. D. Walston, L. P. Fried, A. Singleton, J. Guralnik, G. R. Abecasis, S. Bandinelli, D. L. Longo and L. Ferrucci (2010). "A genome-wide association analysis of serum iron concentrations." Blood 115(1): 94-96. Tanno, T., N. V. Bhanu, P. A. Oneal, S. H. Goh, P. Staker, Y. T. Lee, J. W. Moroney, C. H. Reed, N. L. Luban, R. H. Wang, T. E. Eling, R. Childs, T. Ganz, S. F. Leitman, S. Fucharoen and J. L. Miller (2007). "High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin." Nat Med 13(9): 1096-1101. Tanno, T., P. Porayette, O. Sripichai, S. J. Noh, C. Byrnes, A. Bhupatiraju, Y. T. Lee, J. B. Goodnough, O. Harandi, T. Ganz, R. F. Paulson and J. L. Miller (2009). "Identification of
181
TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells." Blood 114(1): 181-186. Taylor, K. M., H. E. Morgan, A. Johnson and R. I. Nicholson (2005). "Structure-function analysis of a novel member of the LIV-1 subfamily of zinc transporters, ZIP14." FEBS Lett 579(2): 427-432. Theisen, M., R. R. Behringer, G. G. Cadd, R. L. Brinster and G. S. McKnight (1993). "A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice." Mol Cell Biol 13(12): 7666-7676. Tomatsu, S., K. O. Orii, R. E. Fleming, C. C. Holden, A. Waheed, R. S. Britton, M. A. Gutierrez, S. Velez-Castrillon, B. R. Bacon and W. S. Sly (2003). "Contribution of the H63D mutation in HFE to murine hereditary hemochromatosis." Proc Natl Acad Sci U S A 100(26): 15788-15793. Trinder, D., O. Zak and P. Aisen (1996). "Transferrin receptor-independent uptake of differic transferrin by human hepatoma cells with antisense inhibition of receptor expression." Hepatology 23(6): 1512-1520. Trinder, D. and E. Morgan (1998). "Mechanisms of ferric citrate uptake by human hepatoma cells." Am J Physiol 275(2 Pt 1): G279-286. Trinder, D., J. K. Olynyk, W. S. Sly and E. H. Morgan (2002). "Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse." Proc Natl Acad Sci U S A 99(8): 5622-5626. Trinder, D., C. Fox, G. Vautier and J. K. Olynyk (2002). "Molecular pathogenesis of iron overload." Gut 51(2): 290-295. Troadec, M. B., D. M. Ward, E. Lo, J. Kaplan and I. De Domenico (2010). "Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux." Blood 116(22): 4657-4664. Truty, J., R. Malpe and M. C. Linder (2001). "Iron prevents ferritin turnover in hepatic cells." J Biol Chem 276(52): 48775-48780. Turi, J. L., X. Wang, A. T. McKie, E. Nozik-Grayck, L. B. Mamo, K. Crissman, C. A. Piantadosi and A. J. Ghio (2006). "Duodenal cytochrome b: a novel ferrireductase in airway epithelial cells." Am J Physiol Lung Cell Mol Physiol 291(2): L272-280. Valore, E. V. and T. Ganz (2008). "Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin." Blood Cells Mol Dis 40(1): 132-138. Vecchi, C., G. Montosi, K. Zhang, I. Lamberti, S. A. Duncan, R. J. Kaufman and A. Pietrangelo (2009). "ER stress controls iron metabolism through induction of hepcidin." Science 325(5942): 877-880. Verga Falzacappa, M. V., M. Vujic Spasic, R. Kessler, J. Stolte, M. W. Hentze and M. U. Muckenthaler (2007). "STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation." Blood 109(1): 353-358.
182
Videla, L. A., V. Fernandez, G. Tapia and P. Varela (2003). "Oxidative stress-mediated hepatotoxicity of iron and copper: role of Kupffer cells." Biometals 16(1): 103-111. Vokurka, M., J. Krijt, K. Sulc and E. Necas (2006). "Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis." Physiol Res 55(6): 667-674. Wallace, D. F., L. Summerville, P. E. Lusby and V. N. Subramaniam (2005). "First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin." Gut 54(7): 980-986. Wallace, D. F., L. Summerville, E. M. Crampton, D. M. Frazer, G. J. Anderson and V. N. Subramaniam (2009). "Combined deletion of Hfe and transferrin receptor 2 in mice leads to marked dysregulation of hepcidin and iron overload." Hepatology 50(6): 1992-2000. Wallace, D. F., C. J. McDonald, L. Ostini and V. N. Subramaniam (2011). "Blunted hepcidin response to inflammation in the absence of Hfe and transferrin receptor 2." Blood 117(10): 2960-2966. Wallander, M. L., E. A. Leibold and R. S. Eisenstein (2006). "Molecular control of vertebrate iron homeostasis by iron regulatory proteins." Biochim Biophys Acta 1763(7): 668-689. Wang, C. Y. and M. D. Knutson (2013). "Hepatocyte divalent metal-ion transporter-1 is dispensable for hepatic iron accumulation and non-transferrin-bound iron uptake in mice." Hepatology. Wang, J., G. Chen and K. Pantopoulos (2005). "Nitric oxide inhibits the degradation of IRP2." Mol Cell Biol 25(4): 1347-1353. Wang, L., E. E. Johnson, H. N. Shi, W. A. Walker, M. Wessling-Resnick and B. J. Cherayil (2008). "Attenuated inflammatory responses in hemochromatosis reveal a role for iron in the regulation of macrophage cytokine translation." J Immunol 181(4): 2723-2731. Wang, R. H., C. Li, X. Xu, Y. Zheng, C. Xiao, P. Zerfas, S. Cooperman, M. Eckhaus, T. Rouault, L. Mishra and C. X. Deng (2005). "A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression." Cell Metab 2(6): 399-409. Weiss, G., T. Houston, S. Kastner, K. Johrer, K. Grunewald and J. H. Brock (1997). "Regulation of cellular iron metabolism by erythropoietin: activation of iron-regulatory protein and upregulation of transferrin receptor expression in erythroid cells." Blood 89(2): 680-687. Weiss, G. and L. T. Goodnough (2005). "Anemia of chronic disease." N Engl J Med 352(10): 1011-1023. West, A. P., Jr., M. J. Bennett, V. M. Sellers, N. C. Andrews, C. A. Enns and P. J. Bjorkman (2000). "Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE." J Biol Chem 275(49): 38135-38138. West, A. P., Jr., A. M. Giannetti, A. B. Herr, M. J. Bennett, J. S. Nangiana, J. R. Pierce, L. P. Weiner, P. M. Snow and P. J. Bjorkman (2001). "Mutational analysis of the transferrin
183
receptor reveals overlapping HFE and transferrin binding sites." J Mol Biol 313(2): 385-397. Wright, T. L., P. Brissot, W. L. Ma and R. A. Weisiger (1986). "Characterization of non-transferrin-bound iron clearance by rat liver." J Biol Chem 261(23): 10909-10914. Wrighting, D. M. and N. C. Andrews (2006). "Interleukin-6 induces hepcidin expression through STAT3." Blood 108(9): 3204-3209. Xia, Y., J. L. Babitt, Y. Sidis, R. T. Chung and H. Y. Lin (2008). "Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin." Blood 111(10): 5195-5204. Xiong, S., H. She, H. Takeuchi, B. Han, J. F. Engelhardt, C. H. Barton, E. Zandi, C. Giulivi and H. Tsukamoto (2003). "Signaling role of intracellular iron in NF-kappaB activation." J Biol Chem 278(20): 17646-17654. Xiong, S., H. She, C. K. Sung and H. Tsukamoto (2003). "Iron-dependent activation of NF-kappaB in Kupffer cells: a priming mechanism for alcoholic liver disease." Alcohol 30(2): 107-113. Yamaji, S., P. Sharp, B. Ramesh and S. K. Srai (2004). "Inhibition of iron transport across human intestinal epithelial cells by hepcidin." Blood 104(7): 2178-2180. Yang, F., X. B. Liu, M. Quinones, P. C. Melby, A. Ghio and D. J. Haile (2002). "Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation." J Biol Chem 277(42): 39786-39791. Yang, J., D. Goetz, J. Y. Li, W. Wang, K. Mori, D. Setlik, T. Du, H. Erdjument-Bromage, P. Tempst, R. Strong and J. Barasch (2002). "An iron delivery pathway mediated by a lipocalin." Mol Cell 10(5): 1045-1056. Yeh, K. Y., M. Yeh and J. Glass (2004). "Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat." Am J Physiol Gastrointest Liver Physiol 286(3): G385-394. Young, S. P., A. Bomford and R. Williams (1984). "The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes." Biochem J 219(2): 505-510. Zakin, M. M. (1992). "Regulation of transferrin gene expression." Faseb J 6(14): 3253-3258. Zetterstrom, M., A. K. Sundgren-Andersson, P. Ostlund and T. Bartfai (1998). "Delineation of the proinflammatory cytokine cascade in fever induction." Ann N Y Acad Sci 856: 48-52. Zhang, D., E. Meyron-Holtz and T. A. Rouault (2007). "Renal iron metabolism: transferrin iron delivery and the role of iron regulatory proteins." J Am Soc Nephrol 18(2): 401-406. Zhang, D. L., T. Senecal, M. C. Ghosh, H. Ollivierre-Wilson, T. Tu and T. A. Rouault (2011). "Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts." Blood 118(10): 2868-2877.
184
Zhang, Y., M. Mikhael, D. Xu, Y. Li, S. Soe-Lin, B. Ning, W. Li, G. Nie, Y. Zhao and P. Ponka (2010). "Lysosomal proteolysis is the primary degradation pathway for cytosolic ferritin and cytosolic ferritin degradation is necessary for iron exit." Antioxid Redox Signal 13(7): 999-1009. Zhao, M., F. Antunes, J. W. Eaton and U. T. Brunk (2003). "Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis." Eur J Biochem 270(18): 3778-3786. Zhao, N., J. Gao, C. A. Enns and M. D. Knutson (2010). "ZRT/IRT-like protein 14 (ZIP14) promotes the cellular assimilation of iron from transferrin." J Biol Chem 285(42): 32141-32150. Zheng, D., G. P. Feeney, P. Kille and C. Hogstrand (2008). "Regulation of ZIP and ZnT zinc transporters in zebrafish gill: zinc repression of ZIP10 transcription by an intronic MRE cluster." Physiol Genomics 34(2): 205-214. Zhou, X. Y., S. Tomatsu, R. E. Fleming, S. Parkkila, A. Waheed, J. Jiang, Y. Fei, E. M. Brunt, D. A. Ruddy, C. E. Prass, R. C. Schatzman, R. O'Neill, R. S. Britton, B. R. Bacon and W. S. Sly (1998). "HFE gene knockout produces mouse model of hereditary hemochromatosis." Proc Natl Acad Sci USA 95(5): 2492-2497. Zohn, I. E., I. De Domenico, A. Pollock, D. M. Ward, J. F. Goodman, X. Liang, A. J. Sanchez, L. Niswander and J. Kaplan (2007). "The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease." Blood 109(10): 4174-4180.