Mechanisms of Mild Cellular Stress Response Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universitat¨ Berlin by Annabell Plauth (M. Sc.) from Erfurt May 2016 Die Dissertation wurde in der Zeit von Dezember 2011 bis Mai 2016 im Max-Planck- Institut fur¨ molekulare Genetik in Berlin in der Arbeitsgruppe Nutrigenomics and Gene Regulation unter der Leitung von Dr. Sascha Sauer angefertigt. 1. Gutachter: Dr. Sascha Sauer (Max-Planck-Institut fur¨ molekulare Genetik, Universitat¨ Wurzburg)¨ 2. Gutachter: Prof. Dr. Sigmar Stricker (Freie Universitat¨ Berlin) Disputation am: 11.10.2016 To my beloved husband. Acknowledgements I am grateful to my advisor Dr. Sascha Sauer for giving me the opportunity to freely develop my research study on resveratrol, for supporting me with guidance and discus- sions, and for reviewing this thesis. Additionally, I thank Prof. Dr. Sigmar Stricker for reviewing this work. I would like to express my gratitude to my colleagues for their technical support, scien- tific discussions and daily encouragements. My special thanks are to Luise Fuhr, Morten Rousseau, Sophia Bauch, Dr. Christopher Weidner and Dr. Susanne Cichon. I also thank all the other members of the Nutrigenomics and Gene Regulation group for their help and the good working atmosphere. I am further grateful to Dr. Radmila Feldmann, Dr. David Meierhofer, Anja Frei- wald, Silvina Lotito, Linda J. Wainwright for fruitful discussions. Moreover, I wish to acknowledge Sylvia Wowro, Linda Liedgens, and Anne Geikowski. My special thanks are to my dear husband Max, to my family and friends (especially Aylin) for their encouragement, sympathy, and love. Furthermore, I would like to thank the Operating Systems and Middleware Group at Hasso Plattner Institute. This work was supported by Unilever and the Max Planck Society. Contents 1 Introduction1 1.1 The human skin.............................. 2 1.2 Polyphenols................................ 4 1.3 Resveratrol ................................ 7 1.3.1 Chemical properties and stability................. 7 1.3.2 Molecular targets......................... 10 1.3.3 Hormesis ............................. 13 1.4 Reactive oxygen species ......................... 15 1.4.1 Origins .............................. 15 1.4.2 Defense mechanisms....................... 17 1.4.3 Oxidative damage and cellular signaling............. 20 1.5 Nrf2.................................... 21 1.5.1 Regulatory mechanisms of Nrf2 ................. 22 1.5.2 Nrf2-Keap1 signaling: Nexus of metabolic pathways . 26 1.5.3 Protection of the skin....................... 31 1.6 Redox state and redox environment.................... 32 1.7 Aims of this thesis............................. 37 2 Methods and Materials 39 2.1 Compounds and natural products..................... 40 2.2 Decay of resveratrol............................ 40 2.3 Cell culture ................................ 41 2.4 Hematoxylin and eosin staining...................... 44 2.5 Knockdown of Nrf2 and SIRT1...................... 44 2.6 Gene expression analysis......................... 44 CONTENTS 2.7 Genome-wide gene expression analyses ................. 46 2.8 Viability assay............................... 46 2.9 Measurement of reactive oxygen species................. 47 2.10 Antioxidant capacity ........................... 49 2.11 Metabolic parameter measurements.................... 50 2.12 Immunoblotting.............................. 56 2.13 Detection of lipid peroxidation...................... 59 2.14 Fluorescence microscopy......................... 59 2.15 Statistical analyses ............................ 60 2.16 Equipment and reagents.......................... 60 3 Results 65 3.1 Chemical properties of resveratrol .................... 66 3.1.1 Effect of pH............................ 68 3.1.2 Effect of oxygen ......................... 71 3.2 Generation of reactive oxygen species .................. 72 3.2.1 Hydroxyl radical and superoxide generation........... 72 3.2.2 Antioxidant capacity ....................... 74 3.2.3 Antioxidants as scavengers.................... 75 3.3 Cytotoxic effects ............................. 76 3.4 Changes in whole-genome gene expression ............... 78 3.5 Identification of marker genes....................... 81 3.5.1 Inflammation ........................... 81 3.5.2 Proliferation and Autophagy................... 84 3.5.3 Oxidative stress.......................... 90 3.5.4 Energy metabolism........................ 95 3.6 Redox environment............................ 99 4 Discussion 102 4.1 Oxidation of resveratrol..........................103 4.1.1 pH-dependent oxidation .....................104 4.1.2 Oxygen-dependent oxidation...................105 4.2 Resveratrol and reactive oxygen species . 106 CONTENTS 4.2.1 Generation of ROS........................106 4.2.2 Anti- versus pro-oxidant properties . 107 4.3 Hormetic effects of resveratrol caused by oxidation products . 108 4.4 Nrf2 as central mediator..........................110 4.4.1 Inflammatory signaling......................110 4.4.2 Autophagy versus Senescence ..................111 4.4.3 Energy metabolism........................113 4.4.4 Oxidative stress signaling.....................115 4.5 Shift of redox state and environment...................116 4.6 Proposed mechanism of action......................121 4.7 Outlook and future perspectives......................123 5 Summary 125 6 Zusammenfassung 126 7 Bibliography 128 8 Publications 166 9 Supplementary Data 167 9.1 Supplementary Figures ..........................168 9.2 Supplementary Tables...........................178 9.3 Supplementary Equations.........................186 Abbrevations 187 List of Figures 193 List of Tables 195 List of Equations 195 1 Introduction Contents 1.1 The human skin............................ 2 1.2 Polyphenols.............................. 4 1.3 Resveratrol............................... 7 1.3.1 Chemical properties and stability............... 7 1.3.2 Molecular targets....................... 10 1.3.3 Hormesis ........................... 13 1.4 Reactive oxygen species........................ 15 1.4.1 Origins ............................ 15 1.4.2 Defense mechanisms ..................... 17 1.4.3 Oxidative damage and cellular signaling........... 20 1.5 Nrf2................................... 21 1.5.1 Regulatory mechanisms of Nrf2 ............... 22 1.5.2 Nrf2-Keap1 signaling: Nexus of metabolic pathways . 26 1.5.3 Protection of the skin..................... 31 1.6 Redox state and redox environment.................. 32 1.7 Aims of this thesis........................... 37 1 1. INTRODUCTION 1.1 The human skin 1.1 The human skin Covering an area of 1:6 − 1:8 m2 (BioNumbers ID (BNID) 100578 [1]), the human skin is the largest organ of our body [2]. It protects muscles and internal organs from environmental effectors, mediating protection against pathogens and water loss. The skin is responsible for various functionalities, including heat regulation, sensation, excretion (e.g. of sweat), deposition of lipids, and production of vitamin D. Notably, our skin comprises roughly 1:1 ∗ 1011 cells (BNID 101734) covered with approximately 10 bacteria per cell, resulting in a total of 1012 bacteria (BNID 105712 [1]). There are two distinct skin types, thin and thick skin, each comprising three layers called epidermis, dermis, and hypodermis (Figure 1.1). 1.1.1 Layers of the skin The epidermis is the outermost layer of the skin (Figure 1.1) and responsible for the protection against environmental stressors such as pathogens, heat, ultraviolet (UV) radiation, and water loss. As an avascular tissue, the epidermis is sustained solely by diffused oxygen [3]. The epidermis can be divided into five sublayers (bottom to top): stratum basale, spinosum, granulosum, licidum and stratum corneum. Notably, 95% of epidermal cells are keratinocytes (BNID 103639 [1]). During differentiation, keratinocytes produce increased amounts of keratin resulting in a process called keratinization. Consequently, a differentiation gradient with the youngest cells at the bottom (stratum basale) and the oldest, cornified keratinocytes (stratum corneum) at the top is formed. In humans, this keratinocyte turnover takes between 26 and 27 days (BNID 109215 [1]). In total, 1:76 ∗ 1011 cells [4] make up the epidermis, including keratinocytes, melanocytes (causing skin pigmentation), antigen- presenting immune cells, and “touch” cells. 2 1. INTRODUCTION 1.1 The human skin Thick skin (hairless) Thin skin (hairy) Hair shaft Opening of sweat duct Epidermis Papillary dermis Reticular dermis Meissner's Arrector pili muscle corpuscle Dermis Sweat duct Sebaceous gland Subcutis/ Subcutaneous fat hypodermis Hair follicle Eccrine sweat duct Dermal nerve fibres Eccrine sweat gland Eccrine sweat gland Pacinian corpuscle Figure 1.1: Layers of the skin. The human skin contains two skin types, namely thick and thin skin, each containing three layers called epidermis, dermis, and hypodermis. Adapted from [5]. The dermis is the second layer of the skin (Figure 1.1) consisting of connective tissue and providing touch (Meissner’s corpuscle) and heat sensation through about 4 ∗ 106 free nerve endings (BNID 101736 [1]). The dermis is segregated into two sublayers called papillary and reticular region. The latter is characterized by a dense network of matrix components, e.g. collagen, elastin and an extrafibrillar matrix, providing strength and elasticity. This layer