Role and Regulation of the Metalloproteinase ADAM8 in Liver Inflammation and Hepatocellular Carcinoma Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Tanzeela Awan M. Phil Pharmacology aus Layyah, Pakistan Berichter: Herr Universitätsprofessor Dr. rer. nat. Andreas Ludwig Frau Universitätsprofessorin Dr. phil. nat. Gabriele Pradel Herr Universitätsprofessor Dr. rer. nat. Martin Zenke Tag der mündlichen Prüfung: 11.01.2021 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. Table of Contents Table of Contents 1 Introduction .......................................................................................... 1 1.1 The Liver .............................................................................................. 1 1.1.1 Cells of the liver ................................................................................... 1 1.1.2 Liver inflammation ............................................................................... 3 1.1.3 Liver carcinoma .................................................................................... 6 1.1.4 Integrin and focal adhesion kinase signalling ...................................... 7 1.2 Metalloproteinases ................................................................................ 9 1.2.1 ADAM proteases: structure and functions of different domains ....... 10 1.3 ADAM8 .............................................................................................. 12 1.3.1 Expression and activation of ADAM8 ............................................... 12 1.3.2 Pro-inflammatory role of ADAM8 ..................................................... 13 1.3.3 Anti-inflammatory role of ADAM8 ................................................... 15 1.3.4 ADAM8 in cancers ............................................................................. 16 2 Aim of the study ................................................................................. 20 2.1 Role of ADAM8 in liver inflammation .............................................. 20 2.2 Role of ADAM8 in malignancy of HCC ............................................ 21 3 Materials and Methods ....................................................................... 22 3.1 Materials ............................................................................................. 22 3.1.1. Chemicals, enzymes and recombinant proteins ................................. 22 3.1.2 Consumables ...................................................................................... 23 3.1.3 Kits ..................................................................................................... 24 3.1.4 Microorganisms .................................................................................. 24 3.1.5 Plasmids .............................................................................................. 24 3.1.6 Antibodies .......................................................................................... 25 3.1.7 Buffers and solutions .......................................................................... 26 3.1.8 Oligonucleotides ................................................................................. 28 3.1.9 shRNA sequence ................................................................................ 29 3.1.10 Cell lines ............................................................................................. 30 3.2 Methods .............................................................................................. 31 i Table of Contents 3.2.1 Cell culture methods ........................................................................... 31 3.2.2 Protein biochemistry methods ............................................................ 34 3.2.3 Molecular biology methods ................................................................ 37 3.2.4 Functional Assays ............................................................................... 42 3.2.5 Animal experiments ........................................................................... 43 3.3 Statistical analysis .............................................................................. 45 4 Results ................................................................................................ 46 4.1 ADAM8 mediates non-alcoholic fatty liver disease .......................... 46 4.1.1 ADAM8 in NAFLD tissues ................................................................ 46 4.1.2 Expression of ADAM8 in hepatic cell lines ....................................... 48 4.1.3 Regulation of ADAM8 expression in liver cell lines under NAFLD conditions ........................................................................................... 50 4.1.4 Influence of ADAM8 knockdown on liver inflammation by regulating expression and release of cytokines in liver cells ............................... 55 4.2 Role of ADAM8 in LPS-induced liver injury .................................... 72 4.2.1 Regulation of ADAM8 in different liver injury models ..................... 72 4.2.2 ADAM8 and cytokines are regulated by LPS treatment in liver cells in- vitro .................................................................................................... 73 4.2.3 ADAM8 knockout does not protect against LPS induced liver inflammation in-vivo .......................................................................... 79 4.3 Role of ADAM8 in HCC metastasis .................................................. 86 4.3.1 ADAM8 is overexpressed in HCC tissues and hepatoma cell lines ... 87 4.3.2 ADAM8 expression is positively associated with PCNA expression and hepatoma cell proliferation and clonogenicity ................................... 88 4.3.3 ADAM8 expression negatively correlates with caspase 3/7 activity in hepatoma cell lines ............................................................................. 94 4.3.4 ADAM8 expression positively links with cellular migration and invasion of hepatoma cells ................................................................. 96 4.3.5 ADAM8 controls cell proliferation, migration, and angiogenic properties of endothelial cells ........................................................... 100 4.3.6 ADAM8 expression is linked to increased β1 integrin expression and focal adhesion kinase activation ....................................................... 103 4.3.7 ADAM8 is associated with activation of MAPK, Src kinase and Rho A 106 ii Table of Contents 5 Discussion ........................................................................................ 110 5.1 ADAM8 is upregulated during experimental in vitro and in vivo models of NAFLD/NASH and contributes to the release of pro-inflammatory cytokines and fibrotic processes by liver cells in vitro .................... 111 5.2 The up-regulation of ADAM8 by LPS is linked to increased inflammatory mediator production by liver cells but is not critical for LPS-induced liver inflammation in-vivo. ......................................... 117 5.3 ADAM8 critically controls the proliferation and migration in hepatoma cells by regulating the malignant signalling events .......................... 122 5.3.1 ADAM8 regulates cell proliferation, cell migration, cell invasion and apoptosis with HCC metastasis and angiogenesis ............................ 123 5.3.2 ADAM8 is associated with β1 integrin, FAK, Src kinase and Rho A activation in hepatoma cells ............................................................. 126 5.4 Conclusion ........................................................................................ 129 5.5 Outlook ............................................................................................. 131 6 Summary .......................................................................................... 133 7 Literature .......................................................................................... 138 8 Appendix .......................................................................................... 158 8.1 List of figures ................................................................................... 158 8.2 List of tables ..................................................................................... 161 8.3 Abbreviations ................................................................................... 162 8.4 Vector maps ...................................................................................... 166 8.5 Mice genotyping ............................................................................... 170 9 Publications ...................................................................................... 171 10 Curriculum Vitae .............................................................................. 172 11 Declaration ....................................................................................... 173 12 Acknowledgment .............................................................................. 174 iii Introduction 1 Introduction 1.1 The Liver The liver is the body’s largest internal organ, weighing approximately 1500 g and accounting about 2.5 % of the adult body’s weight. It is located at the right upper quadrant of the abdomen and protected by the thoracic cage and diaphragm. The liver is supplied with blood by two major blood vessels: the hepatic artery from the heart and the hepatic portal vein from the gastrointestinal