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The Importance of Inherited and Acquired Keratin Alterations in Liver Disease

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The Importance of Inherited and Acquired Keratin Alterations in Liver Disease Department of Internal Medicine I, University Hospital Ulm Prof. Dr. Seufferlein The importance of inherited and acquired keratin alterations in liver disease Dissertation presented to the Medical Faculty of Ulm University to obtain the degree Doctor of Human Biology Nurdan Güldiken From Erzincan, Turkey 2014 Current Dean: Prof. Dr. Thomas Wirth Thesis reviewers: 1st reviewer: PD Dr. Pavel Strnad 2nd reviewer: Prof. Dr. biol. hum. Uwe Knippschild Date of doctorate awarded: 06.06.2014 To my parents and Ümit, TABLE OF CONTENTS ABBREVIATIONS………………………………………………………………….…...….IV 1. INTRODUCTION ....................................................................................................1 1.1. CYTOSKELETON AND INTERMEDIATE FILAMENTS ........................................................1 1.2. KERATINS-IFS OF EPITHELIAL CELLS ...........................................................................4 1.3. KERATIN EXPRESSION IN HEALTH AND DISEASES.........................................................4 1.4. MECHANICAL AND NON-MECHANICAL FUNCTION OF KERATINS ..................................5 1.5. POSTTRANSLATIONAL MODIFICATIONS OF KERATINS...................................................6 1.6. KERATINS-ASSOCIATED PROTEINS AND FUNCTIONS.....................................................6 1.7. KERATINS AS STRESS INDUCIBLE PROTEINS .................................................................7 1.8. TRANSGENIC ANIMALS AND HUMAN ASSOCIATION STUDIES REVEAL IMPORTANCE OF KERATINS IN THE LIVER ...............................................................................................7 1.8.1. ANIMAL STUDIES .........................................................................................................7 1.8.2. HUMAN STUDIES..........................................................................................................9 1.9. KERATINS EXPRESSION AND DYSREGULATION IN HUMAN LIVER DISEASES ................12 1.10. MECHANISMS OF ACETAMINOPHEN-INDUCED LIVER INJURY......................................12 1.11. ROLE OF KERATIN VARIANTS IN METABOLIC DISORDERS AND LIVER FIBROSIS...........13 AIMS OF STUDY…………………………………………………………...……………………...15 2. MATERIAL AND METHODS……………………………………………....…..16 2.1. MATERIALS ..............................................................................................................16 2.1.1. CHEMICALS AND REAGENTS ......................................................................................16 2.1.2. INSTRUMENTS AND EQUIPMENTS ...............................................................................19 2.1.3. CONSUMABLES ..........................................................................................................20 2.1.4. REACTION KITS AND ENZYMES ..................................................................................21 2.1.5. ANTIBODIES USED FOR WESTERN BLOT AND IMMUNOFLUORESCENCE .......................22 2.1.5.1. PRIMARY ANTIBODIES ...............................................................................................22 2.1.5.2. SECONDARY ANTIBODIES...........................................................................................23 2.1.6. CELL LINES................................................................................................................23 2.1.7. OLIGONUCLEOTIDS (PRIMERS) ..................................................................................23 2.1.7.1. PRIMERS USED FOR GENOTYPING AND SEQUENCING OF HUMAN K8 OVEREXPRESSING MICE GENOM..............................................................................................................24 2.1.7.2. PRIMERS USED FOR QRT-PCR ...................................................................................24 2.1.8. ANIMALS ...................................................................................................................25 2.1.9. BUFFERS....................................................................................................................26 2.2. METHODS .................................................................................................................27 2.2.1. HUMAN SUBJECTS......................................................................................................27 2.2.2. ANIMAL STUDIES .......................................................................................................29 2.2.2.1. ACETAMINOPHEN INDUCED LIVER DAMAGE ..............................................................30 2.2.2.2. BILE DUCT LIGATION (BDL)......................................................................................30 2.2.2.3. HIGH-FAT DIET ..........................................................................................................30 2.2.3. CELL CULTURE EXPERIMENTS....................................................................................31 2.2.4. METHODS OF MOLECULAR BIOLOGY..........................................................................31 2.2.4.1. GENOTYPING OF ANIMALS .........................................................................................31 2.2.4.1.1. DNA ISOLATION........................................................................................................31 2.2.4.1.2. POLYMERASE CHAIN REACTION (PCR)......................................................................31 2.2.4.1.3. GEL ELECTROPHORESIS.............................................................................................32 2.2.4.2. DNA SEQUENCING ....................................................................................................33 2.2.4.3. ISOLATION AND QUANTIFICATION OF RNA................................................................33 2.2.4.4. C-DNA SYNTHESIS FROM RNA .................................................................................34 I 2.2.4.5. QUANTITATIVE REAL TIME PCR (QRT-PCR) ............................................................34 2.2.5. BIOCHEMICAL METHODS............................................................................................36 2.2.5.1. TOTAL PROTEIN EXTRACTION FROM LIVER TISSUE.....................................................36 2.2.5.2. HIGH SALT EXTRACTION OF INSOLUBLE PROTEINS.....................................................36 2.2.5.3. SDS-PAGE AND WESTERN BLOTTING .......................................................................37 2.2.5.4. BRADFORD PROTEIN ASSAY .......................................................................................38 2.2.5.5. HISTOLOGICAL STAININGS.........................................................................................38 2.2.5.5.1. HEMATOXYLIN AND EOSIN (H&E) STAINING ............................................................38 2.2.5.5.2. PICRO-SIRIUS RED STAINING ......................................................................................39 2.2.5.5.3. IMMUNOFLUORESCENCE STAINING ............................................................................39 2.2.5.5.4. OIL-RED STAINING ....................................................................................................40 2.2.5.6. SUBCELLULAR FRACTIONATION ................................................................................40 2.2.5.7. BIOCHEMICAL ASSAYS...............................................................................................41 2.2.5.7.1. TOTAL AND OXIDIZED GLUTATHION (GSH/GSSG) ASSAY .......................................41 2.2.5.7.2. ATP ASSAY ...............................................................................................................41 2.2.5.7.3. TRIGLYCERIDE (TG) ASSAY.......................................................................................42 2.2.5.7.4. MEASUREMENT OF CYTOKINES TNF-A AND IL-6 ......................................................42 2.2.5.7.5. DETERMINATION OF ACETAMINOPHEN-PROTEIN ADDUCTS ......................................43 2.2.6. ANALYSIS AND SOFTWARES.......................................................................................43 2.2.7. ETHICS STATEMENT ...................................................................................................44 2.2.8. SAFETY MEASURES ....................................................................................................44 3. RESULTS................................................................................................................45 3.1. ANALYSIS THE IMPACT OF KERATIN 8 VARIANTS ON LIVER INJURY DEVELOPMENT ..45 3.1.1. K8 VARIANTS EXHIBIT NORMAL LIVER PHENOTYPE UNDER BASAL CONDITIONS ........45 3.1.1.1. BASAL CHARECTERISATION OF K8 TRANSGENIC MICE...............................................45 3.1.1.2. OVEREXPRESSION OF K8 VARIANTS DOES NOT LEAD TO A DISRUPTION OF KERATIN NETWORK ARCHITECTURE. ........................................................................................47 3.1.1.3. KERATIN 8 G62C AND R341C VARIANTS PROMOTE KERATIN 8 CROSSLINKING.........48 3.1.2. KERATIN 8 VARIANTS PREDISPOSE TO ACETAMINOPHEN-TOXICITY ...........................49 3.1.2.1. KERATIN 8 VARIANTS PREDISPOSE TO APAP-INDUCED LIVER INJURY.......................49 3.1.2.2. KERATIN G62C/R341C/H VARIANTS LEAD TO INCREASED ACETAMINOPHEN-INDUCED NECROSIS...................................................................................................................51 3.1.2.3. PRESENCE OF KERATIN G62C/R341C/H VARIANTS DOES NOT AFFECT APAP METABOLISM .............................................................................................................53 3.1.2.4. INCREASED
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