STUDIES ON THE CYTOPROTECTIVE ROLE OF AUTOPHAGY IN NECROSIS WU YOUTONG (M. Med, Huazhong Univ Sci & Tech, P. R. CHINA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF EPIDEMIOLOGY AND PUBLIC HEALTH NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to dedicate my sincere and deep gratitude to my supervisor, Associate Professor Shen Han-Ming, for his enthusiastic professional guidance. Prof. Shen has led me into the vast world of biological research and has guided me with inspirations during the tough, yet exciting, journey throughout this study. Besides acting as a mentor, Prof. Shen has also treated me like a friend, sharing with me of his invaluable experience in life philosophy, communication skills, and even protocols for delicious cooking. All of these will be appreciated in my whole life. I would also like to express my sincere thanks to Prof. Ong Choon Nam, who offered me the precious opportunity for working in this lab in Singapore, which fueled my dream to pursue this Ph.D degree. It has been a great pleasure for me to work in the big family of Department of Epidemiology and Public Health in the last four years. All people in the lab are always kind and helpful. I would like to list the members of this big family with honor and gratitude: Mr. Ong Her Yam, Dr. Zhang Siyuan, Dr. Huang Qing, Dr. Lu Guo-Dong, Dr. Zhou Jing, Ms. Su Jin, and Mr. Ong Yeong Bing. Finally, I would like to express my deep appreciation to my wife, Ms Tan Hui-Ling. Without her contribution of diligent and elegant works to this study and her dedicated love and understanding, this thesis would not be possible. II TABLE OF CONTENTS ACKNOWLEDGEMENTS II TABLE OF CONTENTS III SUMMARY VI LIST OF TABLES IX LIST OF FIGURES X LIST OF ABBREVIATIONS XII LIST OF PUBLICATIONS XV CHAPTER INTRODUCTION 1.1 PROGRAMMED CELL DEATH: APOPTOSIS VERSUS NECROSIS 1.1.1 APOPTOSIS 1.1.2 NECROSIS 1.1.3 CROSSTALK BETWEEN APOPTOSIS AND NECROSIS 1.2 AUTOPHAGY 1.2.1 GENERAL INTRODUCTION 1.2.2 DYNAMIC PROCESS OF AUTOPHAGY 1.2.3 MACHINERY FOR AUTOPHAGOSOME FORMATION 1.2.4 REGULATORY PATHWAYS 1.2.5 AUTOPHAGY FUNCTIONS AND IMPLICATIONS IN DISEASES 1.3 CROSSTALK BETWEEN AUTOPHAGY AND PCD 1.3.1 AUTOPHAGY IN APOPTOSIS 1.3.2 AUTOPHAGY IN NECROSIS 1.4 OBJECTIVES 14 19 21 21 22 24 31 42 46 47 53 60 CHAPTER AUTOPHAGY HAS A PROTECTIVE ROLE DURING ZVAD-INDUCED NECROTIC CELL DEATH 62 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 2.2.1 REAGENTS AND ANTIBODIES 2.2.2 CELL CULTURE AND TREATMENTS 2.2.3 DETECTION OF CELL DEATH 2.2.4 TRANSIENT TRANSFECTION AND CONFOCAL MICROSCOPY ANALYSIS 2.2.5 ELECTRON MICROSCOPY ANALYSIS 63 65 65 66 66 66 67 III 2.2.6 SIRNA 67 2.2.7 MEASUREMENT OF CATHEPSIN B ACTIVITY 67 2.2.8 WESTERN BLOTTING 68 2.3 RESULTS 69 2.3.1 ZVAD INDUCES CASPASE-INDEPENDENT NON-APOPTOTIC CELL DEATH WITH THE PRESENCE OF AUTOPHAGY MARKERS IN L929 CELLS 69 2.3.2 OPPOSITE EFFECTS OF RAPAMYCIN AND CHLOROQUINE ON ZVAD-INDUCED CELL DEATH 70 2.3.3 OPPOSITE EFFECT OF SERUM STARVATION AND ATG GENE KNOCKDOWN ON ZVAD-INDUCED CELL DEATH 71 2.3.4 ZVAD SUPPRESSES LYSOSOMAL FUNCTION VIA ITS INHIBITORY EFFECT ON CATHEPSIN ACTIVITY 72 2.3.5 ZVAD INHIBITS AUTOPHAGOSOME MATURATION 73 2.4 DISCUSSION 74 CHAPTER ACTIVATION OF THE PI3K-AKT-MTOR SIGNALING PATHWAY BY INSULIN PROMOTES NECROTIC CELL DEATH VIA SUPPRESSION OF AUTOPHAGY 91 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 3.2.1 REAGENTS AND ANTIBODIES 3.2.2 CELL CULTURE 3.2.3 DETECTION OF CELL DEATH/CELL VIABILITY 3.2.4 PLASMIDS AND STABLE TRANSFECTION 3.2.5 CONFOCAL MICROSCOPY 3.2.6 SMALL INTERFERING RNA 3.2.7 WESTERN BLOTTING 3.3 RESULTS 3.3.1 INSULIN PROMOTES CELL DEATH IN NECROTIC CELL DEATH MODELS 3.3.2 INSULIN ABOLISHES THE PROTECTIVE EFFECT OF STARVATION ON NECROTIC 92 94 94 95 95 95 96 96 96 97 97 98 99 CELL DEATH 3.3.3 IGF-1, BUT NOT EGF, HAS A SIMILAR PRO-DEATH EFFECT AS INSULIN 3.3.4 INHIBITION OF PI3K-AKT-MTOR SIGNALING PATHWAY BY CHEMICAL INHIBITORS ABOLISHES THE PRO-DEATH EFFECT OF INSULIN 3.3.5 KNOCKDOWN OF MTOR MITIGATES THE PRO-DEATH EFFECT OF INSULIN 3.3.6 INSULIN INHIBITS AUTOPHAGY INDUCED BY STARVATION 3.4 DISCUSSION 100 101 102 103 CHAPTER ZVAD-INDUCED NECROPTOSIS IN L929 CELLS DEPENDS ON AUTOCRINE PRODUCTION OF TNFΑ MEDIATED VIA THE PKC-MAPKS-AP-1 PATHWAY 119 4.1 INTRODUCTION 120 IV 4.2 MATERIALS AND METHOD 122 4.2.1 REAGENTS AND ANTIBODIES 122 4.2.2 CELL CULTURE 123 4.2.3 DETECTION OF CELL DEATH 123 4.2.4 SIRNA 123 4.2.5 TRANSFECTION AND LUCIFERASE REPORTER ASSAY 124 4.2.6 REVERSE TRANSCRIPTION-PCR 124 4.2.7 MEASUREMENT OF AUTOCRINE TNFΑ IN CULTURE MEDIUM BY ELISA 125 4.2.8 WESTERN BLOTTING 125 4.3 RESULTS 125 4.3.1 ZVAD AND BOCD, BUT NOT QVD, INDUCE NECROSIS IN L929 CELLS 125 4.3.2 ZVAD-INDUCED NECROTIC CELL DEATH REQUIRES DE NOVO PROTEIN SYNTHESIS 127 4.3.3 ZVAD-INDUCED CELL DEATH IS RIP1- AND RIP3-DEPENDENT 127 4.3.4 ZVAD PROMOTES AUTOCRINE PRODUCTION OF TNFΑ 128 4.3.5 BLOCKAGE OF TNFΑ SIGNALING SUPPRESSES ZVAD-INDUCED NECROPTOSIS 129 4.3.6 NF-ΚB PATHWAY IS NOT INVOLVED IN ZVAD-INDUCED AUTOCRINE PRODUCTION OF TNFΑ, BUT PLAYS A PROTECTIVE ROLE DURING ZVAD-INDUCED NECROPTOSIS 130 4.3.7 AP-1 ACTIVITY IS REQUIRED FOR ZVAD-INDUCED TNFΑ PRODUCTION AND CELL DEATH 131 4.3.8 ZVAD-INDUCED AP-1 ACTIVATION IS MEDIATED BY JNK AND ERK 132 4.3.9 PKC PLAYS A CRITICAL ROLE IN ZVAD-MEDIATED MAPKS-AP1 ACTIVATION, TNFΑ PRODUCTION, AND CELL DEATH 133 4.3.10 ZVAD SENSITIZES TNFΑ-INDUCED NECROPTOSIS IN L929 CELLS 135 4.3.11 DEFECT IN AUTOPHAGY ENHANCES AP-1 ACTIVITY 135 4.4 DISCUSSION 137 CHAPTER GENERAL DISCUSSIONS AND CONCLUSIONS 163 5.1 AUTOPHAGY PLAYS A PRO-SURVIVAL ROLE IN ZVAD-INDUCED NECROSIS 5.2 ZVAD INHIBITS AUTOPHAGY VIA SUPPRESSION OF CATHEPSIN ACTIVITY 5.3 SUPPRESSION OF AUTOPHAGY BY ACTIVATION OF PI3K-AKT-MTOR AXIS 165 166 5.4 AUTOCRINE TNFΑ IS THE DEATH SIGNAL IN ZVAD-INDUCED NECROSIS 5.5 DUAL ROLE OF ZVAD DURING INDUCTION OF NECROPTOSIS 5.6 THE MECHANISMS FOR AUTOPHAGY TO PROTECT NECROSIS 5.7 CONCLUSIONS 168 170 173 176 178 CHAPTER REFERENCES 181 PROMOTES NECROSIS V SUMMARY Programmed cell death (PCD) is an intrinsically regulated cellular suicide process that can be categorized into apoptosis and necrosis based on their distinct morphological characteristics. Autophagy refers to an evolutionarily conserved process that sequesters and targets bulk cellular constituents for lysosomal degradation. Autophagy has been found to be implicated in regulation of PCD under various cellular settings. At present, the role of autophagy on PCD is highly controversial. Although autophagy generally serves as a cell survival mechanism under stress conditions such as starvation, there are reports showing that autophagy executes caspase-independent cell death, known as autophagic cell death. However, in many cases the evidence supporting autophagy as a cell death mechanism is frequently circumstantial and appears inadequate. zVAD, a pan-caspase inhibitor, has been shown to induce robust necrosis in L929 cells, and such necrosis has been reported as autophagic cell death. However, the molecular mechanism underlying such cell death has not been fully elucidated. Therefore, the main objective of this study is to investigate the regulatory role of autophagy in necrosis and to elucidate the underlying molecular mechanisms using in vitro mammalian cell models. The following investigations have been conducted: (i) examining the role of autophagy in zVAD-induced necrosis by modulation of autophagy via either pharmacological or genetic approaches; (ii) studying the regulatory role of class I PI3K-Akt-mTOR signaling axis in modulation of autophagy and necrosis; and (iii) elucidating the molecular mechanism underlying zVAD-induced necrosis. VI In this study, we first demonstrated that autophagy played a cytoprotective role during zVAD-induced necrosis. Moreover, zVAD was able to suppress autophagy via suppression of lysosome function via inhibition of cathepsin enzyme activity. One surprising finding of this study was that growth factors such as insulin and IGF-1 and nutrients such as amino acids were able to enhance zVAD-induced necrosis via activation of the PI3K-Akt-mTOR pathway and subsequent suppression of autophagy. Moreover, the pro-death function of insulin/amino acids was also observed in other two necrosis models, including MNNG-induced necrosis in L929 cells and H2O2-induced necrosis in Bax/Bak double knockout cells, where autophagy acted as a pro-survival mechanism. Finally, we identified that zVAD-induced necrosis was RIP1- and RIP3-mediated necroptosis that depended on the autocrine production of TNFα. zVAD promoted the autocrine production of TNFα at the transcription level, which was required for induction of cell death. We also demonstrated that zVAD promoted TNFα production via the PKC-MAPKs-AP-1 pathway. Moreover, we presented evidence showing that defects in autophagy might promote zVAD-induced cell death by enhancing AP-1 activity. In conclusion, data from this study demonstrate that (i) autophagy plays a cell survival strategy in the three necrosis models tested in this study; (ii) growth factors and amino acids promote necrosis in these models via activation of the PI3K-Akt-mTOR pathway and subsequent suppression of autophagy; and (iii) zVAD-induced necroptosis depends on autocrine production of TNFα that is mediated via the PKC-MAPKs-AP-1 signaling pathway. Taken together, results from the VII above-described studies provide novel insights for a better understanding of the role of autophagy in necrosis. VIII LIST OF ABBREVIATIONS 3-MA 4EBP1 ActD AIF AMPK ANT Apaf-1 Atg ATP BAFF Bak Bax Bcl-2 Beclin BH BNIP3 BocD-fmk CARD CHX CNS CQ DAPK DED DEVD-cho DISCs DR DRAM DUB EAA EBSS EGF EGFP eIF2α ER ERK FADD FBS FIP200 fmk FoxO FoxOs GAPDH 3-methyladenine eukaryotic initiation factor 4E binding protein actinomycin D apoptosis-inducing factor AMP-activated protein kinase adenine nucleotide translocase apoptotic peptidase activating factor-1 autophagy-related adenosine triphosphate B-cell activating factor Bcl-2 homologous antagonist Bcl-2 associated X protein B-cell lymphoma-2 coiled-coil, myosin-like BCL2 interacting protein Bcl-2 homology BCL2 and adenovirus E1B 19 kDa-interacting protein Boc-Asp(Ome)-fmk caspase recruitment domain cycloheximide central nervous system chloroquine death-associated protein kinase death effector domain Asp-Glu-Val-Asp-cho death-inducing signaling complexes death receptor damage-regulated autophagy modulator deubiquitinating enzyme essential amino acid earle’s balanced salt solution epidermal growth factor green fluorescent protein eucaryotic translation initiation factor 2α endoplasmic reticulum extracellular signal-regulated kinases Fas-associated death domain fetal bovine serum focal adhesion kinase family interacting protein of 200 Kd fluoromethylketone forkhead box O forkhead box O transcription factors glyceraldehyde-3-phosphate dehydrogenase IX GDP GLUD1 GLUL GTP HIF1 hVps IAP ICAD IETD-fmk IETD-oph IGF-1 IKK IL IκB JNK KO LC3 LPS MAPK Mcl-1 Mdm MEF MNNG MOMP mRFP mTOR mTORC1 mTORC2 MTT NAD NADPH NF-κB NIK Nox1 oph PAR PARP PAS PCD PDK1 PE PERK PH PI PI3K Guanosine diposphate glutamate dehydrogenase glutamate ammoniavligase Guanosine triposphate hypoxia-induced factor human vacuolar protein sorting inhibitor of apoptosis inhibitor of caspase activated DNase or DNA fragmentation factor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fmk Z-Ile-Glu(OMe)-Thr-Asp(OMe)-oph insulin-like growth factor-1 IκB kinase interleukin Inhibitor of κB c-Jun N-terminal kinase knockout rat microtubule-associated protein light chain lipopolysaccharide mitogen-activated protein kinase Myeloid cell leukemia sequence-1 murine double minute mouse embryonic fibroblast Methylnitronitrosoguanidine mitochondrial outer membrane permeabilization monomeric red fluorescent protein mammalian target of rapamycin mTOR complex mTOR complex 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide β-nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate hydrogen nuclear factor kappa-light-chain-enhancer of activated B cells NF-κB-inducing kinase NADPH oxidase 2,6-difluorophenoxy methyl Ketone poly(ADP-ribose) poly(ADP-ribose) polymerase pre-autophagosome structure programmed cell death phosphoinositide-dependent kinase phosphatidylethanolamine PKR-like ER kinase pleckstrin homology propidium iodide phosphoinositide-3 kinase X Liang, C., Feng, P., Ku, B., Dotan, I., Canaani, D., Oh, B.H., and Jung, J.U. 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Genes Dev 20, 1-15. 213 [...]... effect of starvation on zVAD-induced necrotic cell death in L929 cells Figure 3.3 Other growth factors and amino acids have a similar pro-death effect as insulin Figure 3.4 Inhibition of the PI3K activity abolishes the pro-death effect of insulin on zVAD-induced necrosis in L929 cells Figure 3.5 Inhibition of mTOR activity by rapamycin abolishes the pro-death effect XIII of insulin Figure 3.6 Knockdown of. .. protective role in zVAD-induced cell death Figure 4.6 Knockdown of c-Jun blocks zVAD-induced autocrine TNFα production and necroptosis Figure 4.7 zVAD-induced TNFα transcription is depending on the MAPKs-AP-1 signaling pathway Figure 4.8 Critical role of PKC in zVAD-induced MAPKs-AP-1 activation, autocrine of TNFα and necroptosis Figure 4.9 Promotion of autocrine of TNFα combining with caspase-8 inhibition induces... bind to and antagonize functions of the anti-apoptosis Bcl-2 proteins to promote apoptosis (Danial, 2007) The Bcl-2 family members are predominantly involved in regulation of the intrinsic apoptotic pathway The fundamental underlying mechanism is that the Bax and Bak are able to induce the mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c for initiating the intrinsic... 2008) According to the length of their prodomains and the positions in the apoptotic signaling cascade, caspases can be classified into two groups, the initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10, -11, -12) and the effector caspases (caspase-3, -6, -7) Initiator caspases harbor long prodomains containing a protein-protein interaction motif, the death effector domain (DED) or the caspase... such machineries have been established, the extrinsic and the intrinsic pathways The extrinsic apoptotic pathway is classically initiated by the cell death receptors, such as TNF receptor 1 (TNFR1), Fas, and death receptor (DR) 4/5 The engagement of the cell death ligands with their respective receptors induces the formation of intracellular death-inducing signaling complexes (DISCs) consisting of multiple... mitigates the pro-death effect of insulin on necrosis Figure 3.7 Insulin suppresses autophagy induced by starvation Figure 4.1 Caspase inhibition is not sufficient for zVAD to induce necrosis Figure 4.2 zVAD-induced necrosis requires de novo protein synthesis and depends on RIP1 and RIP3 Figure 4.3 zVAD promotes autocrine of TNFα Figure 4.4 Blockage of TNF signaling pathway prevents zVAD-induced cell... implying that these two types of necrosis may utilize distinct routes for JNK activation and the consequent cell demise Interestingly, JNK has also been found to act upstream of PARP-1 and contributes to sustained PARP-1 activation, leading to necrosis in response to oxidative stress (Zhang et al., 2007) Therefore, the relationship and potential crosstalk between these two types of necrosis remain to... susceptible to necroptosis when engineered with RIP3 (Zhang et al., 2009) However, whether RIP3 also performs similar functions in PARP-1-mediated necrosis is not known These findings may provide novel insights and directions for us to further investigate the key role of caspase in necrotic cell death Interestingly, in addition to being capable of switching apoptosis into necrosis, 20 ... N-(2-Quinolyl)valyl-aspartyl-oph regulatory associated protein of mTOR V-rel reticuloendotheliosis viral oncogene homolog Ras homolog enriched in brain rapamycin-insensitive companion of mTOR receptor interacting protein reactive oxygen species p70 S6 kinase short interfering RNA second mitochondria-derived activator of caspase transactivation domain truncated Bid tandem fluorescent-tagged LC3 construct... its functions (Wahl, 2006) Besides, activation of p53 is governed by a variety of post-translational modifications (PTMs) including acetylation, phosphorylation, methylation, poly(ADP-ribosyl)ation et al For example, aceylation of lysine 373 of p53 by p300 and/or CBP markedly increases its transactivities toward the lower affinity-binding target genes (Knights et al., 2006) Phosphorylation of p53 at . elucidate the underlying molecular mechanisms using in vitro mammalian cell models. The following investigations have been conducted: (i) examining the role of autophagy in zVAD-induced necrosis. SUPPRESSION OF AUTOPHAGY BY ACTIVATION OF PI3K-AKT-MTOR AXIS PROMOTES NECROSIS 168 5.4 AUTOCRINE TNFΑ IS THE DEATH SIGNAL IN ZVAD-INDUCED NECROSIS 170 5.5 DUAL ROLE OF ZVAD DURING INDUCTION OF NECROPTOSIS. to suppress autophagy via suppression of lysosome function via inhibition of cathepsin enzyme activity. One surprising finding of this study was that growth factors such as insulin and IGF-1