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Inhibitors of Caspase-1 Function 21 III ACTIVATION OF CASPASE-1 SIGNALING COMPLEXES BY THE P2X7 RECEPTOR REQUIRES INTRACELLULAR K+ EFFLUX AND PROTEIN SYNTHESIS INDUCED BY PRIMING WITH TOLL-LIKE RECEPTOR LIGANDS by JOANNE MICHELLE KAHLENBERG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. George R. Dubyak Department of Pathology CASE WESTERN RESERVE UNIVERSITY August, 2004 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. I would like to dedicate this work to my husband, Mark. Thank you for your friendship, for your support, and for all the times you told me to suck it up and just do it. I love you. TABLE OF CONTENTS LIST OF FIGURES 3 ACKNOWLEDGEMENTS 5 LIST OF ABBREVIATIONS 6 ABSTRACT 7 CHAPTER 1. Introduction I. IL-1β is a highly regulated cytokine that requires a secondary stimulus for cleavage and release IL-1β, the protein 10 IL-1β secretion 13 mIL-1β formation 14 II. The activation of caspase-1 is required for the cleavage of IL-1β Caspase-1, the protein 16 Activators of caspase-1 function 17 Inhibitors of caspase-1 function 21 III. The activation of caspase-1 involves the assembly of multimeric complexes The CARD domain 23 The apoptosome 24 The inflammasome 27 Detailed analysis of inflammasome components 30 Negative regulators of inflammasome assembly 36 IV. P2X7 is an important receptor for blood and neuronal tissues Pharmacology and physiology of P2 Receptors 37 P2X7R can signal in a variety of cell types 41 P2X7R downstream signaling 43 P2X7R activation induces IL-1β processing and release 46 Inhibitors of P2X7R-mediated IL-1β processing 47 V. Toll-like Receptors 48 CHAPTER 2. General Methods 51 1 CHAPTER 3. Differing Caspase-1 Activation States in Monocyte Versus Macrophage Models of IL-1β Processing and Release Summary 60 Introduction 61 Results 63 Discussion 76 CHAPTER 4 Mechanisms of Caspase-1 Activation by P2X7 Receptor-Mediated K+ Release Summary 82 Introduction 83 Results 85 Discussion 98 CHAPTER 5 Potentiation of Caspase-1 Activation by the P2X7 Receptor is Dependent on Toll Like Receptor Signals and Involves NFκB-Driven Protein Synthesis Summary 105 Introduction 106 Results 107 Discussion 124 CHAPTER 6 Discussion and Future Directions Does P2X7R activation produce isolatable inflammasome complexes? 131 What is the role for nucleotides in inflammasome assembly? 138 Is there a role for the lysosome in P2X7R-mediated caspase-1 activation? 140 Does the c-terminal tail of P2X7R recruit inflammasome or NFκB activating complexes? 144 BIBLIOGRAPHY 147 2 LIST OF FIGURES Figure 1.1. IL-1β and caspase-1 are synthesized in pro forms that require cleavage for activation………………………………………………12 Figure 1.2 The apoptosome assembles using the CARD domains of scaffolding proteins and caspase-9……………………………...…..25 Figure 1.3 The inflammasome assembles using CARD and PYRIN domains…..29 Figure 1.4 The P2X7R is an ATP-gated ion channel that induces pore formation with prolonged stimulation…………………………..…..40 Figure 3.1 THP-1 monocytes but not Bac1 macrophages release large amounts of mIL-1β in response to LPS stimulation…………..64 Figure 3.2 Rates of caspase-1 and IL-1β processing differ in cell-free lysates from Bac1 macrophages versus THP-1 monocytes…………………66 Figure 3.3 THP-1 lysate components can accelerate caspase-1 and IL-1β processing in Bac1 lysates…………………………………………..69 Figure 3.4 Transactivation of processing reactions in Bac1 lysates is dependent on heat-labile THP-1 lysate factors >10 kDa……………71 Figure 3.5 Active caspase-1 heterotetramers can induce accelerated processing in Bac1 cell-free lysates…………………………………73 Figure 3.6 Constitutive caspase-1 activation in THP-1 lysates is insensitive to inhibition by AG126 or BEL………………………………………...75 Figure 4.1 Bac1 macrophages release IL-1β after 30 min, even with only a 5 min ATP pulse…………………………………………………...86 Figure 4.2 ATP stimulation induces changes within intact Bac1 macrophages that accelerate caspase-1 activation in vitro…………………………88 Figure 4.3 Acceleration of in vitro processing requires >1 mM ATP Stimulation…………………………………………………………..89 Figure 4.4 Stimulus-induced acceleration of caspase-1 activation is dependent on K+ loss from intact cells……………………………92 Figure 4.5 AG126 and BEL inhibit ATP-induced acceleration of IL-1β processing without affecting K+ release……………………...94 Figure 4.6 mASC accelerates caspase-1 and IL-1β processing in Bac1 lysates…………………………………………………………97 Figure 4.7 The activation of P2X7R results in the activation of caspase-1 by stable protein complexes…………………………………………………...101 Figure 5.1 Activation of caspase-1 by the P2X7R requires priming with LPS…………………………………………………………….110 Figure 5.2 Activation of caspase-1 by P2X7R and nigericin requires LPS priming………………………………………………………....111 3 Figure 5.3 LPS priming of caspase-1 activation by the P2X7R can be mimicked by TLR2 and TLR9 signals………………………114 Figure 5.4 LPS priming of caspase-1 activation by the P2X7R does not require ERK, JNK, p38 or PI3K pathways………………..117 Figure 5.5 LPS priming of caspase-1 activation by P2X7R activation is dependent on protein synthesis………………...…………………120 Figure 5.6 LPS priming of caspase-1 activation by the P2X7R requires the activity of the proteasome……………………………………….123 Figure 6.1 TLR signals upregulate factors required for P2X7R-mediated caspase-1 activation…………………………………………………132 Figure 6.2 Caspase-1, IL-1β, and caspase-12 shift into higher molecular weight fractions with incubation at 30°C………………..133 Figure 6.3 ATP stimulation of Bac-1 macrophages induces recruitment of procaspase-1 and IL-1β to a ~2MDa complex……………………...135 Figure 6.4 ATP treatment can induce association of caspase-1 with IL-1β and possibly Nalp1……………………………………...137 Figure 6.5 Activated charcoal inhibits the spontaneous processing of IL-1β, but processing cannot be restored upon ATP stimulation………......139 4 ACKNOWLEDGEMENTS I would first like to thank my mentor, George Dubyak, for all that he has done for me. George’s enthusiasm for science is truly infectious. I learned much about science from George, but more importantly, I hope to take away the lessons in integrity and concern for people that he demonstrates on a day-to-day basis. I would also like to thank the Dubyak lab members for helping me when I needed it and for providing a fun work environment. Special thanks go to Sylvia Kertesy for her experimental assistance. Thanks also to the former members of the Templeton lab who helped me to gain skills in molecular biology. My gratitude also extends to my first and to my current thesis committees. Their advice and support have been invaluable. I would also like to thank my parents who have always trusted my decisions and have given me their full support and love. I have to give a big thank you to Amelia Sutton for her support and encouragement when times were bad and good. Thanks also to all my friends and family who have supported me and provided me with much needed distractions. Lastly, I would like to thank my husband, Mark, for everything he does. 5 LIST OF ABBREVIATIONS BAPTA 1,2-bis(2-Aminophenosy)ethane-N,N,N’N’- tetraacetic acid tetrakis(acetoxymethyl ester) BEL Bromoenol Lactone BzATP 3’-O-(4-benzoyl)benzoyl-ATP CARD Caspase Recruitment Domain CATERPILLER CARD, transcription enhancer, R-binding, pyrin, lots of leucine repeats CINCA Chronic Neurologic Cutaneous and Articular syndrome CLL Chronic Lymphocytic Leukemia COP CARD-Only Protein CRID Cytokine Release Inhibitory Drug FMF Familial Mediterranean Fever FPLC Fast Protein Liquid Chromatography HPLC High Performance Liquid Chromatography ICE Interleukin-1 Converting Enzyme IFNγ Interferon gamma IKK IκB Kinase IL-1β Interleukin-1β IL-1Ra IL-1 Receptor Antagonist LPS Lipopolysaccharide MDP LD Muramyl Dipeptide LD conformation mIL-1β Mature IL-1β ++ iPLA2 Ca-independent Phospholipase A2 PAMP Pathogen-Associated Molecular Pattern PAPA Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne PI3K Phosphoinositol-3 Kinase PI9 Proteinase Inhibitor 9 P2X7R P2X7 Receptor Serpin Serine Protease Inhibitors TCA Trichloroacetic Acid TIR Toll and IL-1 Receptor TLR Toll-like Receptor TNFα Tumor Necrosis Factor alpha 6 Activation of Caspase-1 Signaling Complexes by the P2X7 Receptor Requires Intracellular K+ Efflux and Protein Synthesis Induced by Priming with Toll-Like Receptor Ligands Abstract By JOANNE MICHELLE KAHLENBERG Recent advances in understanding of the biology underlying hereditary inflammatory diseases have focused attention on the importance of regulating levels of the proinflammatory cytokine IL-1β to prevent systematic inflammation. Diseases such as Muckle-Wells Syndrome and Familial Mediterranean Fever are characterized by inflammatory arthritis, episodic fevers and rashes, and amyloid deposition as well as elevated serum levels of IL-1β. These inflammatory symptoms can be eliminated by antagonists to the IL-1β receptor, stressing the role of elevated IL-1β levels in inducing these inflammatory phenotypes. Additionally, the identification of genetic
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