MECHANISMS OF RIBOTOXIC STRESS RESPONSE AND DOWNSTREAM SEQUELAE By Kaiyu He A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Microbiology - Environmental Toxicology 2012 ABSTRAC MECHANISMS OF RIBOTOXIC STRESS RESPONSE AND DOWNSTREAM SEQUELAE By Kaiyu He Translational inhibitors and other translation-interfering toxicants, termed ribotoxins, activate MAPKs via a process termed ribotoxic stress response (RSR). Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium spp. , is a ribotoxin and commonly contaminates cereal-based foods and has the potential to adversely affect humans and animals. At low doses, DON induces immunostimulatory effects by upregulating expression of proinflammatory genes in macrophages, IL-8 in monocytes and IL-2 in T cells. In contrast, high doses of DON cause immunosuppression by inducing apoptosis and rRNA cleavage. While it is recognized that DON induces transcription and stability of inflammation-associated mRNAs in the macrophage, it is not known whether this toxin can selectively modulate translation of these mRNAs. DON-induced changes in profiles of polysome-associated mRNA transcripts (translatome) was compared to total cellular mRNA transcripts (transcriptome) in the RAW 264.7 murine macrophage model. DON induced robust expression changes in inflammatory response genes including cytokines, cytokine receptors, chemokines, chemokine receptors, and transcription factors, which were remarkably similar in the translatome and transcriptome. Over 70 percent of DON- regulated genes in the translatome and transcriptome overlapped and most expression ratios in these pools are <2. Taken together, DON’s capacity to alter translation expression of inflammation-associated genes is likely to be driven predominantly by selective transcription, however, a small subset of these genes might further be regulated at the translational level. The complete cleavage profile and exact signaling mechanism of DON-induced rRNA cleavage are unknown. PKR, Hck and p38 were found to be required for rRNA cleavage. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-µ as well as the pan caspase inhibitor Z-VAD-FMK. DON activated caspases 3, 8 and 9 thus suggesting the possible co-involvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Notably, pan inhibitor for cathepsins also suppressed anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Accordingly, all four ribotoxins induced apoptosis-associated rRNA cleavage via activation of cathepsins and p53 →caspase 8/9 →caspase 3, the activation of which by DON and anisomycin involved PKR-and Hck-activated p38 whereas SG and ricin activated p53 by an alternative mechanism. Taken together, at low doses, DON selectively upregulates translation of inflammation-associated genes, which is likely to be driven predominantly by selective transcription of these genes. However, a small subset of these genes might further be regulated at the translational level. At high doses, DON induces apoptosis-associated rRNA cleavage via activation of cathepsins and PKR/Hck/p38/p53 →caspase 8/9→caspase 3. Interestingly, DON and anisomycin share the same signaling pathways, whereas SG and ricin activate p53 by an alternative mechanism, indicating the downstream signalings are conserved for ribotoxins. ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor, Dr. James Pestka, for his excellent guidance, patience, and providing an excellent atmosphere for doing research at Michigan State. He helped me to develop various skills, such as independent thinking and scientific writing, from which I will benefit not only for my future researches but also my whole life. I am grateful to my Committee members: Dr. Robert Britton, Dr. Kathleen Gallo and Dr. John Linz for their invaluable suggestions, assistance and guidance. I am also thankful to all the members in Dr. Pestka and Dr. Linz’s laboratories, especially Dr. Hui- Ren Zhou, who, as a good friend and scientific mentor, was always willing to help me and give me best suggestions. I would also like to thank my parents, younger brother and wife, Qi Wang. They were always supporting me and encouraging me with their best wishes. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... vii LIST OF FIGURES ........................................................................................................ viii LIST OF ABBREVIATIONS .............................................................................................xi INTRODUCTION ............................................................................................................. 1 CHAPTER 1.Literature Review ....................................................................................... 4 A.Trichothecenes ......................................................................................................... 5 B. Deoxynivalenol (DON) ............................................................................................. 6 C. Mitogen-activated Protein Kinases (MAPKs) ........................................................... 9 D. Ribotoxic stress response ..................................................................................... 11 E. Apoptosis ............................................................................................................... 26 F. Translational regulation.......................................................................................... 30 G. Summary ............................................................................................................... 30 CHAPTER 2. Modulation of Inflammatory Gene Expression by the Ribotoxin Deoxynivalenol Involves Coordinate Regulation of the Transcriptome and Translatome .................................................................................................................. 35 ABSTRACT ................................................................................................................ 36 INTRODUCTION ....................................................................................................... 37 MATERIALS AND METHODS ................................................................................... 40 RESULTS .................................................................................................................. 44 DISCUSSION ............................................................................................................. 59 CHAPTER 3. Mechanisms of Ribosomal RNA (rRNA) Cleavage by the Trichothecene DON....................................................................................................... 65 ABSTRACT ................................................................................................................ 66 INTRODUCTION ....................................................................................................... 67 MATERIALS AND METHODS ................................................................................... 70 RESULTS .................................................................................................................. 77 DISCUSSION ............................................................................................................. 96 v CHAPTER 4. Mechanisms for Ribotoxin-induced Ribosomal RNA Cleavage ............. 101 ABSTRACT .............................................................................................................. 102 INTRODUCTION ..................................................................................................... 104 MATERIALS AND METHODS ................................................................................. 107 RESULTS ................................................................................................................ 110 DISCUSSION ........................................................................................................... 129 CHAPTER 5. Summary and future research ............................................................... 135 APPENDICES ............................................................................................................. 139 Appendix A. Role of PKR in Ribotoxic Stress Response ........................................ 140 Appendix B. Construction and Expression of FLAG-tagged Ribosomal Proteins in HEK 293T and Hela Cells ....................................................................................... 174 Appendix C. Comparison of DON-induced Proinflammatory Gene Expression in Wildtype and PKR Knockout Mice .......................................................................... 195 Appendix D. DON-induced Modulation of MicroRNA Expression in RAW 264.7 Macrophages- A Potential Novel Mechanism for Translational Inhibition. .............. 210 REFERENCES ............................................................................................................ 221 vi LIST OF TABLES Table 2.1. Functional gene grouping of DON-induced up- and down-regulated genes in transcriptome and tranlatome. ................................................................................... 45 Table 2.2. DON-induced up-regulation of inflammatory response genes in translatome (TLM) and transcriptome (TCM). ............................................................... 46 Table 2.3. DON-induced