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Pkr Dependent Upregulation of Immediate Early Genes And PKR DEPENDENT UPREGULATION OF IMMEDIATE EARLY GENES AND ANTI-INFLAMMATORY CYTOKINE IL-10. A dissertation submitted to Kent State University in cooperation with the Cleveland Clinic in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Arindam Chakrabarti May, 2007 Dissertation written by Arindam Chakrabarti M.S., University of Calcutta, 1999 Ph.D. Kent State University, 2007 Approved by ____________________ Chair, Doctoral Dissertation Committee Bryan R. G. Williams, Ph.D ____________________ Member, Doctoral Dissertation Committee Robert H. Silverman, Ph.D ____________________ Member, Doctoral Dissertation Committee Gail Fraizer, Ph.D ____________________ Member, Doctoral Dissertation Committee Jennifer Marcinkiewicz, Ph.D ____________________ Member, Doctoral Dissertation Committee Volodymyr Dvornyk, Ph.D ____________________ Graduate Faculty Representative Arne Gericke, Ph.D Accepted by ____________________ Director, School of Biomedical Sciences Robert V. Dorman, Ph.D ____________________ Dean, College of Arts and Sciences John R. D. Stalvey, Ph.D ii TABLE OF CONTENTS List of Figures ……………………………………………………………………………….v List of Tables………………………………………………………………………….……..vi Acknowledgements …………………………………………………………………..…….vii Chapter 1. Introduction ....…………………………………………………………..……...1 Chapter 2. PKR mediated uregulation of immediate early gene egr-1 Abstract………………………………………………………………………………..…42 Introduction ………………………………………………………………………..…….…..43 Materials and Methods ……………….…………………………………………..…...……..46 Results ………………………………………………………………………………..……...53 Discussion ……………………………………………………………………..……..……...73 Chapter 3. PKR mediated uregulation of immediate early gene c-Jun Abstract………………………………………………………………………………..…75 Introduction ……………………………………………………………………………...…..76 Materials and Methods ……………………………..……………………………..........……79 Results …………………………………………………………………………………….... 83 Discussion ………………………………………………………….…………..……..……103 Chapter 4. PKR mediated uregulation of anti-inflammatory cytokine IL-10 Abstract………………………………………………………………………..………..106 Introduction …………………………………………………………………..…………….107 Materials andMethods ……………………………………………….………………..…....110 Results …………………………………………………………………………………..….115 Discussion ………………………………………………………………………....……….136 Chapter 5. General discussion and Future Direction Summary ………………………………………………………….…………..…….…..….140 Future perspectives ………………………………………….………………………...……145 Appendix A. Abbreviations ………………….……………………..………………...……...158 References …………………………………………………….……………..…..…….163 iv LIST OF FIGURES Chapter 1: Fig 1.1 TLR7,8,9 mediated signaling………………………………………………..……7 Fig 1.2 TLR3 mediated signaling…………………………………………………..……..8 Fig 1.3 RIG-1 mediated signaling………………………………….……………….....…11 Fig 1.4 NFκB signaling pathway……………………………………………………..….35 Chapter 2: Fig 2.1 Characterization of wt and pkr-ko SM cells………………………...………..….54 Fig 2.2 Microarray analysis of pIC treated wt and pkr-ko SM cells….………………….58 Fig 2.3 Validation of microarray result by real time PCR…………………………….....64 Fig 2.4 Induction of Egr-1 protein expression is dependent on PKR…………….......…66 Fig 2.5 Egr-1 induction in response to pIC is dependent on NF-κB……………........….68 Fig 2.6 pIC mediated activation of Egr-1 promoter is dependent on NF-κB…….....…...71 Chapter 3: Fig 3.1 PKR dependent induction of c-Jun………………………………………..….…85 Fig 3.2 pIC mediated c-Jun induction is dependent on JNK and Erk1/2………………...89 Fig 3.3 pIC mediated activation of MAP kinases is impaired in absence of PKR…...….93 Fig 3.4 Impaired activation of MKK4 in absence of PKR……………………….……...98 Fig 3.5 c-Jun promoter activity is induced in response to pIC……………………...…...99 Fig 3.6 CREB is involved in pIC driven c-Jun induction………………………….…...101 Chapter 4: Fig 4.1 PKR dependent IL-10 induction……………………………………………..…116 Fig 4.2 IL-10 induction is dependent on JNK and NF-κB………….………………..…119 Fig 4.3 Activation of JNK and NF-κB is dependent on PKR…………….………..…...125 Fig 4.4 Luciferase assay using a mouse IL-10 promoter fragment………………..……128 Fig 4.5 Binding of p65 and p50 to the IL-10 promoter in vivo…………………..……..130 Fig 4.6 IL-10 induction in response to viral infection…………………………….……132 Fig 4.7 pIC mediated IL-10 induction causes STAT3 activation.………………….…..134 Chapter 5: Fig 5.1 pIC mediated tristetraprolin induction is largely dependent on NF-κB and partly onp38……………………………………………………………………………..…..…151 Fig 5.2 pIC mediated activation of CREB is dependent on JNK………………...…..…152 Fig 5.3 PKR dependent upregulation of pro-inflammatory genes……………….…......153 Fig 5.4 Model………………………………………………………………...………....158 v LIST OF TABLES Table 1. PKR dependent upregulation of genes……………………………...…………..74 vi ACKNOWLEDGEMENT For the greater part of my work I have been encouraged and guided by many whose contribution it may not be possible to individually acknowledge here. But, even within that large list, it would be impossible not to mention those who have made an immense imprint. My parents have always been a constant source of encouragement, strength and support, and my dreams of becoming what I am today would have remained distant had they not, in turn, had their own dreams and vision on their son. Were I to single out one person for all that I achieved over the last few years, it would undoubtedly be Dr Bryan Williams, without whom, all my efforts would have been in vain. Extremely supportive, a great motivator and most generous in a student’s pursuit of knowledge he will, and always will remain, my ideal forever. I am deeply indebted to him. I sincerely thank my distinguished committee members Dr.Robert Silverman, Dr. Gail Fraizer, Dr. Jennifer Marcinkiewicz and Dr Volodomyr Dvornyk for their invaluable guidance at different stages of my work. Had it not been for them it would have been impossible to walk the last mile. vii During my research I have been privileged to have worked alongside Tony, Mark, Joao, Jeanna to name a few, who gave their abundant support unhesitatingly. It will never be easy to find that spontaneity elsewhere but I will continue to hope that I find such a group of excellent co-workers in future as well. Many of my friends and well-wishers, unnamed here but always a source of strength, have also been indulgent to me and I would like to thank them all for their selfless support. I would like to thank my wife Maupali, whose invaluable support in everything that I did or couldn’t, whose avid interest and eager inquisitions on my subject, whose never ending effort to free me so that I could fully engage myself in my work made all the difference between success and failure. I have to admit that, like all aspiring students, I have faced pangs of dejection and disappointments. Even during those phases, the mirth and endless chatter of a three year old child, with tiny hands extended in warmth, helped me quickly recover and get back to the task in hand. Love you son. viiixi Chapter 1 Introduction Vertebrates are constantly threatened by the invasion of microorganisms and have evolved systems of immune defense to effectively eliminate infective pathogens in the body. The mammalian immune system comprises of innate and acquired immunity. The innate immune system is the first line of host defense against pathogens and is mediated by phagocytes including macrophages and dendritic cells (DCs). Acquired immunity,on the other hand is involved in elimination of pathogens in the late phase of infection and in generation of immunological memory. Activation of antiviral innate immune response relies on detection of pathogen associated molecular patterns (PAMPS), which includes viral DNA, single-stranded RNA and double-stranded RNA (dsRNA). Members of Toll-like receptor family TLR9 and TLR7/8 recognizes unmethylated CpG DNA motifs, present in bacterial or viral DNA and viral single stranded RNA respectively. Whereas, double-stranded RNA (dsRNA), a well known molecular intermediate generated during viral replication of many viruses is recognized by a number of cellular proteins like, TLR3, PKR, RNA helicases like RIG-I and Mda-5. Cumulatively, these host components are called pattern recognition receptos (PRR). Following recognition of viral components the PRRs promote production of variety of cytokines, activation of several physiological processes and induction of innate 1 and adaptive immune responses critical for generating effective and appropriate antiviral response. Toll-like receptor dependent antiviral pathways : TLRs are evolutionarily conserved from the worm C. elegans to mammals (Akira and Takeda, 2004; Beutler, 2004; Hoffmann, 2003; Janeway and Medzhitov, 2002). 12 members of TLR family have been identified to date. Toll-like receptors are type I integral membrane glycoproteins which have variable numbers of leucine-rich-repeat (LRR) motifs in their extracellular domains and a conserved cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/IL-1R homology (TIR) domain (Bowie et al., 2000). TLRs are expressed on various immune cells, including macrophages, dendritic cells (DCs), B cells, specific types of T cells, and even on nonimmune cells such as fibroblasts and epithelial cells. Expression of TLRs is modulated rapidly in response to pathogens, a variety of cytokines, and environmental stresses. TLRs can reside at extracellular or intracellular locations. Interestingly, TLRs that are involved in recognition of different nucleic acid and observed to participate in recognition of viral infection (TLR3, TLR7/8 and TLR9) have been
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