ALEJANDRA RODRÍGUEZ MARTÍNEZ Expression Profiling of Novel Iron-Related Genes in Mouse Models of Iron Overload ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine of the University of Tampere, for public discussion in the Small Auditorium of Building B, Medical School of the University of Tampere, Medisiinarinkatu 3, Tampere, on October 30th, 2009, at 12 o’clock. UNIVERSITY OF TAMPERE ACADEMIC DISSERTATION University of Tampere, Institute of Medical Technology Tampere Graduate School in Biomedicine and Biotechnology (TGSBB) Finland Supervised by Reviewed by Professor Seppo Parkkila Professor Markku Heikinheimo University of Tampere University of Helsinki Finland Finland Professor Debbie Trinder University of Western Australia Australia Distribution Tel. +358 3 3551 6055 Bookshop TAJU Fax +358 3 3551 7685 P.O. Box 617 [email protected] 33014 University of Tampere www.uta.fi/taju Finland http://granum.uta.fi Cover design by Juha Siro Acta Universitatis Tamperensis 1460 Acta Electronica Universitatis Tamperensis 894 ISBN 978-951-44-7862-8 (print) ISBN 978-951-44-7863-5 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://acta.uta.fi Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2009 CONTENTS TIIVISTELMÄ .....................................................................................................6 ABSTRACT..........................................................................................................8 LIST OF ORIGINAL COMMUNICATIONS....................................................10 ABBREVIATIONS.............................................................................................11 1. INTRODUCTION..........................................................................................14 2. REVIEW OF THE LITERATURE................................................................16 2.1 Body iron homeostasis ............................................................................16 2.1.1 Iron distribution in humans...........................................................16 2.1.2 Intestinal iron absorption..............................................................18 2.1.2.1 Iron transport across the apical mucosal surface..............18 2.1.2.2 Iron export to plasma .......................................................20 2.1.3 The transferrin iron pool...............................................................21 2.1.4 Iron recycling................................................................................22 2.2 Cellular iron metabolism.........................................................................23 2.2.1 Cellular acquisition of iron...........................................................23 2.2.1.1 The transferrin cycle ........................................................23 2.2.1.2 Other means of transferrin-iron uptake............................24 2.2.1.3 Uptake of non-transferrin-bound iron ..............................25 2.2.2 Cellular iron storage .....................................................................26 2.2.3 Cellular iron export.......................................................................26 2.3 Regulation of iron homeostasis ...............................................................27 2.3.1 Regulation of cellular iron homeostasis .......................................27 2.3.2 Regulation of systemic iron homeostasis .....................................28 2.3.2.1 Hepcidin, a negative regulator of iron transport ..............28 2.3.2.2 Regulation of hepcidin expression...................................30 2.4 Iron overload ...........................................................................................32 2.4.1 General..........................................................................................32 2.4.2 Hereditary hemochromatosis........................................................33 2.4.2.1 HH type 1: mutated HFE..................................................34 2.4.2.2 HH type II or juvenile hemochromatosis: mutated hepcidin and hemojuvelin ..................................36 3 2.4.2.3 HH type III: mutated TFR2..............................................38 2.4.2.4 HH type IV: mutated ferroportin .....................................39 2.4.3 Animal models of iron overload...................................................39 3. AIMS OF THE STUDY ................................................................................41 4. MATERIALS AND METHODS...................................................................42 4.1 Protein expression analyses.....................................................................42 4.1.1 Antibody production (I) ...............................................................42 4.1.2 Western blotting (I)......................................................................42 4.1.3 Immunohistochemistry (II) ..........................................................43 4.2 mRNA expression analyses.....................................................................44 4.2.1 Conventional reverse transcription PCR (I).................................44 4.2.1.1 Sequencing of PCR products ...........................................45 4.2.2 Quantitative reverse transcription PCR (II-IV) ...........................46 4.2.2.1 Murine tissue samples......................................................46 4.2.2.2 RNA extraction and cDNA synthesis ..............................46 4.2.2.3 Quantitative reverse transcription PCR ...........................47 4.2.2.4 Statistical analyses (III, IV) ............................................50 4.2.3 cDNA Microarray (III, IV)..........................................................51 4.2.3.1 Experimental procedure...................................................51 4.2.3.2 Data analysis ....................................................................51 4.3 Mouse models of iron overload ..............................................................52 4.3.1 Dietary iron overload (III, IV).....................................................52 4.3.1.1 Determination of tissue iron content and statistical analysis (III) ....................................................53 4.3.2 Hfe-/- mice (IV).............................................................................53 4.4 Ethical approval (II-IV)..........................................................................54 5. RESULTS ......................................................................................................55 5.1 Iron content in the liver and heart of iron-fed mice (III,IV) ..................55 5.2 Expression of Hemojuvelin.....................................................................55 5.2.1 Hemojuvelin mRNA in human and mouse tissues (I, II) ............55 5.2.2 Hemojuvelin protein in mouse tissues (I, II) ...............................56 5.2.3 Hemojuvelin transcript in the heart, skeletal muscle and liver of mice with dietary iron overload (III)...............................57 5.3 Expression of Neogenin..........................................................................58 5.3.1 Neogenin transcript in mouse tissues (II) ....................................58 5.3.2 Neogenin protein in mouse tissues (II) ........................................58 4 5.3.3 Neogenin mRNA in heart, skeletal muscle and liver of mice with dietary iron overload (III) ...........................................59 5.4 Expression of iron-related genes in the heart, skeletal muscle, liver and duodenum of iron-loaded mice (III, IV).................................60 5.4.1 Hepcidin........................................................................................60 5.4.2 Other iron-related genes ...............................................................60 5.5 Expression of iron-related genes in the liver and duodenum of Hfe-/- mice (IV) .......................................................................................61 5.5.1 Hepcidin........................................................................................61 5.5.2 Other iron related genes................................................................61 5.6 Global transcriptional response to dietary iron overload in murine heart and skeletal muscle (III) ...................................................61 5.7 Global transcriptional response to Hfe-/- and dietary iron overload in liver and duodenum of mice (IV)........................................63 5.7.1 Hepatic transcriptional response to Hfe deficiency and dietary iron overload.....................................................................63 5.7.1.1 Confirmation of hepatic microarray results by Q- RT-PCR............................................................................66 5.7.2 Duodenal gene expression response to Hfe deficiency and dietary iron supplementation .................................................66 5.7.2.1 Confirmation of microarray results by Q-RT- PCR ..................................................................................67 6. DISCUSSION ................................................................................................68 6.1 Expression profiles of hemojuvelin and neogenin ..................................68 6.2 The RNA microarray technique ..............................................................70 6.3 Transcriptional changes in the heart and skeletal muscle of iron-loaded mice .....................................................................................70 6.4 Transcriptional changes in the liver of mice with iron