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Cloning, characterization, anti -apoptotic molecular mechanism, transgenic and protein binding studies of mouse TOSO gene

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Cloning, characterization, anti -apoptotic molecular mechanism, transgenic and protein binding studies of mouse TOSO gene

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Content NOTE TO USERS This reproduction is the best copy available. ® UMI R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CLONING, CHARACTERIZATION, ANTI-APOPTOTIC MOLECULAR MECHANISM, TRANSGENIC AND PROTEIN BINDING STUDIES OF MOUSE TOSO GENE Yahui Song A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY (MOLECULAR MICROBIOLOGY AND IMMUNOLOGY) August 2004 Copyright 2004 Yahui Song R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UMI Number: 3145293 INFORM ATION TO USERS The quality of this reproduction is dependent upon the quality of the copy subm itted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and im proper alignm ent can adversely affect reproduction. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if unauthorized copyright m aterial had to be rem oved, a note will indicate the deletion. ® UMI UMI Microform 3145293 Copyright 2004 by ProQuest Inform ation and Learning Company. All rights reserved. This m icroform edition is protected against unauthorized copying under Title 17, United States Code. ProQ uest Inform ation and Learning C om pany 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. DEDICATION To my loving grandfather Caisan Yang for his wisdom, to my loving parents Ruiruo Yang and Yuntian Song for their encouragements, and to my loving brother Zhaohui Song for his belief in me. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ACKNOWLEDGEMENTS I wish to express my deepest appreciation to my advisors, Drs. Chaim Jacob, Michael Lai and Louis DuBeau, for their supports and guidance in making this dissertation possible. I am deeply grateful for their critical and valuable suggestions. I am also indebted to Mr. Wenxue E, Drs. Michael Lai, James Ou, Tu-nun Chang, and John Dreher for their efforts in saving my career during the most difficult time of my studies at USC. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TABLE OF CONTENTS Dedication ii Acknowledgements iii List of Tables and Figures vi Abstract x CHAPTER I: INTRODUCTION 1.1 Overview of Apoptosis 1 1.2 Caspases 2 1.3 Extrinsic signaling pathway 6 1.3.1 Fas/CD95 pathway 7 1.3.2 TRAIL pathway 15 1.3.3 TN pathway 19 1.4 Intrinsic signaling pathway 24 CHAPTER II: CLONING, CHARATERIZATION, FUNCTION AND MOLECULAR MECHANISM STUDIES OF mTOSO 2.1 Abstract 25 2.2 Introduction 27 2.3 Material and Methods 32 2.4 Results 40 2.5 Discussion 63 CHAPTER III: T-CELL SPECIFIC TRANSGENIC STUDIES OF mTOSO 3.1 Abstract 69 3.2 Introduction 71 3.2.1 Autoimmune Diseases 71 3.2.2 TOSO transgenic mice 74 3.3 Material and Methods 76 3.4 Results 79 3.5 Discussion 85 CHAPTER IV: PROTEIN BINDING STUDIES OF mTOSO 4.1 Abstract 87 4.2 Introduction 88 4.3 Material and Methods 94 4.4 Results 96 4.4.1 1-acylglycerol-3-posphate O-acyltransferase 3 96 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 4.4.2 Eukaryotic translation elongation factor 1 beta 2 97 4.4.3 Ca(2+)-transporting ATPase 2C1 98 4.5 Discussion 102 CHAPTER V: CONCLUSION AND FUTURE DIRECTIONS 5.1 Future directions 104 5.2 Conclusions 107 BIBLIO GRAP Y 181 APPENDEX A: INHIBITION OF RNA POLYMERASE I TRANSCRIPTION A.l IN DIFFERENTIATED MYELOID LEUKEMIA CELLS BY INACTIVATION OF SELECTIVITY FACTOR 1 Background 109 A.2 Abstract 114 A. 3 Introduction 115 A.4 Material and Methods 119 A. 5 Results 122 A. 6 Discussion 140 APPENDEX B: MOLECULAR MECHANISM OF DOWN-REGULATION OF NF-kB BY IL-10 B.l Background 145 B.2 Abstract 150 B.3 Introduction 151 B.4 Material and Methods 154 B.5 Results 159 B.6 Discussion 175 v R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. LIST OF TABLES AND FIGURES Table 1: Sequences of microsatellite primers 78 Figure 1-1: Fas/CD95 pathway of apoptosis 10 Figure 1-2: Caspases pathway of apoptosis 11 Figure 1-3: TRAIL pathway of apoptosis 17 Figure 1-4: TNF pathway of apoptosis 23 Figure 2-1A.: Alignment of cDNA sequences of human TOSO and mouse TOSO gene products 41 Figure 2-1B: Alignment of amino acid sequences of human TOSO and mouse TOSO gene products 43 Figure 2-2: Kyte-Doolittle hydropathy plot analysis of human TOSO and mouse TOSO proteins 44 Figure 2-3: The partial alignment of mTOSO amino acid sequences in normal and autoimmune mice strains 45 Figure 2-4: Tissue expression of mouse TOSO mRNA 47 Figure 2-5: Mouse TOSO protein locates on cell membrane 49 Figure 2-6: RT-PCT result of TOSO in stably transfected clones 51 Figure 2-6: Ectopic expression of mouse TOSO protects Jurkat cells from FasL induced apoptosis 52 Figure 2-7: Mouse TOSO protects Jurkat cells from TNFa-induced apoptosis 54 vi R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2-8: Mouse TOSO inhibits caspase-8 activation in Fas-induced apoptosis 55 Figure 2-9: mTOSO binds to FADD 57 Figure 2-10 A: Schematic structure of mTOSO deletion constructs 58 Figure 2-10B: In vitro translation of 3 5S-labeled mTOSO deletions 58 Figure 2-10C: Binding of mTOSO deletions to GST 59 Figure 2-10D: Binding of mTOSO deletions to GST-FADD 59 Figure 2-11: TRAIL treatment of mTOSO transfected Jurkat cells 62 Figure 3-1: The construct of the lck-mTOSO transgene 79 Figure 3-2: Southern blot analysis of mTOSO transgenic mice 80 Figure 3-3: Screening of FI mice from NZW background crossed with mTOSO founder mice #8 and #20 on B6BDAF1 background 81 Figure 3-4A: Realtime PCR of mTOSO transgene expression 83 Figure 3-4B: Primary T lymphocytes from transgenic mice treated with FasL 84 Figure 4-1: Schematic representation of yeast two hybrid system 90 Figure 4-2: Schematic representation of split ubiquitin system 92 Figure A -l: RNA polymerase I transcription decreases after U937 cells differentiation 123 Figure A-2: Extracts from TPA-treated cells are deficient in pol I T ranscription activity 125 vii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure A-3: Differentiated U937 cell extract does not contain a soluble repressor 127 Figure A-4 Undifferentiated and differentiated U937 cells extracts display similar levels of pol I polymerization activity 129 Figure A-5A: Exogenous SL1 can stimulate pol I transcription I differentiated U937 cell extract 130 Figure A-5B: Pol I transcription in differentiated U937 nuclear extracts can be stimulated by the addition of purified SL1 131 Figure A-5C: Transcription activity of Hela-purified UBF 132 Figure A-6A: SL1 activity is repressed in differentiated U937 cell extracts 134 Figure A-6B: UBF activity in U937 cell extracts 135 Figure A-7A: Western blot analysis of TBP, TAFs and UBF from undifferentiated and differentiated U937 cells 137 Figure A-7B: Western blot analysis of TBP from alkaline phosphatase-treated U937 cells 137 Figure A-8: Immunoprecipitation of SL1 from undifferentiated and differentiated U937 extracts 139 Figure B -l: NF-kB DNA-binding activity is inhibited by IL-10 in HL-60 T Cells 161 Figure B-2: IL-10 inhibits NF-kB DNA-binding activity by preventing TNFa Induced degradation of IkB a 161 Figure B-3: RT-PCR analysis of IL10R1 and IL-10R2 expression in different cell lines 164 Figure B-4: IL-10 inhibits NF-kB DNA-Binding Activity through inhibiting TNF-Induced Translocation of I k B 165 Figure B-5: Jakl is not required to inhibit TNF-induced IkB Translocation by IL-10 168 V lll R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure B-6: Tyk2 is not required to inhibit TNF-induced IkB Translocation by IL-10 Figure B-7: Figure B-8: Tyrosines at positions 446 and 496 of IL-10R1 at the Stat docking site are not required to inhibit TNF-induced IkB Translocation by IL-10 28 Amino Acids at the Carboxyl Terminal of IL-10R1 are Required to inhibit TNF-induced IkB Translocation by IL-10 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ABSTRACT Apoptosis or programmed cell death is a genetically encoded physiological form of cell death that plays a critical role in development and tissue homeostasis in multicellular organisms. When deregulated, it can contribute to the pathogenesis of diseases such as cancer, autoimmune disease, viral infection and neurodegenerative disorders. In this study, mouse TOSO gene, the homologue of human TOSO gene which inhibits apoptosis mediated by members of the TNF receptor family, was cloned. Its mRNA expression pattern in various mouse organs was determined and its putative cell membrane subcellular localization was confirmed. To identify the molecular mechanism of how it inhibits apoptosis, TOSO stably transfected cell lines were generated, in vitro protein binding and mutagenesis studies were performed. The results indicate that TOSO inhibits Fas-induced apoptosis by inhibiting caspse-8 activation through its binding to FADD molecule. Accumulating evidence suggests apoptosis is playing a vital role in autoimmune diseases. Studies have suggested that insufficient apoptosis can result in autoimmunity or cancer, whereas accelerated apoptosis leads to degenerative diseases, immunodeficiency and infertility. To study the role played by TOSO in autoimmune diseases, TOSO transgenic mice with its targeted expression in T lymphocytes were generated. TOSO transgenic mice showed earlier onset of the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. autoimmune symptoms, comparing to non-transgenic littermates on lupus genetic background. The analyses of protein interactions taking place in the cells are essential for understanding their functions in the cellular context. Three potential binding proteins of TOSO were identified through split-ubiquitin screening system. Further in vitro and in vivo functional studies of these proteins will yield more insights into how TOSO functions on molecular level. In conclusion, the results yield from this study demonstrated that TOSO is a key regulator in apoptosis and plays an important role in autoimmune diseases. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER 1 INTRODUCTION 1.1 Overview of Apoptosis Apoptosis or programmed cell death is a genetically encoded physiological form of cell death that plays a critical role in development and tissue homeostasis in multicellular organisms. Intact regulation of cell death during embryonic development is essential for successful organogenesis and crafting of complex multicellular tissues. Apoptosis plays an important role in long-lived mammals where multiple signals are integrated and homeostasis is maintained, which is critical in precluding pathogenesis. When deregulated, it can contribute to the pathogenesis of diseases such as cancer, autoimmune disease, viral infection and neurodegenerative disorders (Kidd, 1998). Apoptosis is featured by unique characteristics, including cell shrinkage, plasma membrane blebbing, chromatin condensation, internucleosomal DNA fragmentation, phosphatidylserine exposure and, finally, formation of apoptotic bodies. Unlike necrosis where cells swell and burst, releasing their intracellular contents and causing damage to surrounding cells and inflammation, apoptosis occurs without any sign of inflammation and disruption of tissue structure, and neighboring cells rapidly phagocytose apoptotic bodies (Wyllie et al., 1980). The first clue to the molecular basis of apoptosis came from genetic studies in the nematode Caenorhabditis elegans. The pioneering contributions to development 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. genetics and programmed cell death by Nobel Prize Laureates Brenner, Horvitz and Sulston brought key insights and paved way for the studies in this field (Brenner, 1974; Sulston, 1976; Ellis and Horvitz, 1986). The central components in apoptosis in C. elegans are ced3, ced4 and ced-9 genes. Ced-3 encodes an aspartate-specific cysteine protease of the caspase family which is required for apoptosis to occur in normal development. Ced-4 protein is a proapoptotic adaptor molecule which directly interact with Ced-3 and activate pro-Ced-3. In contrast to Ced-3 and Ced-4, Ced-9 is anti-apoptotic. Ced-9 binds to ced-4 and prevents Ced-4-mediated activation of proCed-3 (Hengartner et al., 1992; Yuan and Horvitz, 1992; Yuan et al., 1993). The mammalian homologues of Ced-9 are the Bcl-2 caspase family members which, along with other caspases, are responsible for the execution of apoptosis in various higher eukaryotes (Gross et al., 1999; Cryns and Yuan, 1998). 1.2 Caspases A central role of regulation and execution of apoptosis is played by a group of cysteine proteases, caspases, which cleave protein substrates at aspartic acid residue (Cryns et al., 1998). The targets of caspases include cell structural or house keeping proteins, DNA repair proteins, nucleases and proteins involved in signal transduction (Nunez et al., 1998). Caspases are constitutively present in the cytosol of most cells as a single proenzyme, known as procaspases, and are activated by upstream caspases or other protease such as granzyme B (Green, 1998). They are activated by the removal of the N-terminal prodomain. The zymogens contain 2 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. prodomain, the p20 large and the pl7 small subunits. To activate procaspases, the large and small subunits are cleaved first, followed by the removal of prodomain (Ramage et al., 1995; Yamin et al., 1996). Currently, 11 human caspases have been identified, including caspase-1-10 and caspase-14 (Alnemri et al., 1996; Pistritto et al., 2002). Caspase-11 and -12 are murine enzymes with homology to human caspase-4 and -5, respectively, whereas caspase-13 is a bovine homologue of caspase-4 (Lamkanfi et al., 2002; Koenig et al., 2001). It is clear that there is an evolutionary tendency to increase the number of caspases over phylogenetic time, from 4 in C. elegans to 7 in Drosophila and 11 in mice and humans (Lamkanfi et al., 2002). Activation of caspases can be blocked by Inhibitors of Apoptosis (IAPs) (LaCasse et al., 1998). Functionally, caspases are grouped into either “initiator” caspases or “executioner” caspases (Salvesen et al., 1997; Villa et al., 1997). The initiator caspases are responsible for processing and activation of the executioners, while the executioners are responsible for the cleavage of many cellular proteins that lead to morphological changes and DNA fragmentation associated with apoptosis. The initiator caspases contain long prodomains and one or both of the characteristic protein-protein interaction domains: the death effector domain (DED) (caspase-8 and -10), and caspase activation and recruitment domain (CARD) (caspase-1, -2, -4, -5, -9, -11 and -12). The initiator caspases link to distinct extra- and intracellular signals that trigger apoptosis. On the other hand, the downstream executioner caspases, which are characterized by their short prodomains, cleave multiple cellular 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. substrates (Thomberry et al., 1997). Caspases can also be classified into different groups according to their substrate specificity. They are separated into three classes based on in vitro peptide-aminomethyl-coumarin (AMC) substrates (Rano et al., 1997; Kang et al., 2000). These two kinds of classification are consistent with each other, in the aspect that the substrate preferences of initiator caspases are the downstream executioner caspases. The structure studies of active caspases-1, -3, -7, -8 and -9 have provided valuable insights into the substrate specificity and catalytic mechanism (Walker et al., 1994; Wilson et al., 1994; Rotonda et al., 1996; Mittl et al., 1997; Blanchard et al., 1999; Watt et al., 1999; Wei et al., 2000; Renatus et al., 2001). The active caspase is a homodimer, with six antiparallel ( 5 strands in each monomer and active center from each monomer locating symmetrically on the opposite sides of the enzyme. The specificity of caspases is predominantly determined by the four amino-acid residues N-terminal to the scissile bond (P4-P1) (Margolin et al., 1997). Caspase cleaves only a very small subset of cellular proteins due to its strict substrate specificity. About 100 substrates have been identified to date (Eamshwa et al., 1999; Li and Yuan, 1999; Utz and Anderson, 2000). They are divided into six major categories: 1) proteins directly involved in the regulation of apoptosis; 2) regulatory proteins in apoptosis signaling transduction, such as protein kinases; 3) structural and essential function proteins; 4) proteins required for cellular repair; 5) regulatory proteins involved in cell cycle; 5) and proteins involved in human pathologies. Thus, 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. caspases also serve as signaling mediators that orchestrate in the interwoven signaling pathways. In addition to the proteolytic regulation of caspases, their activities are further regulated by the IAPs (inhibitors of apoptosis proteins) (Liston et al., 2003). IAPs are a family of proteins that function as intrinsic regulators of caspase cascade. IAPs (Cp-IAP, Op-IAP and Ac-IAP) were first identified from baculoviruses based on their ability to inhibit the apoptotic response of host cells to viral infection. Several cellular homologues of IAPs have been identified such as c-IAP-l/hIAP2/MIHB, c-IAP-2, and NAIP. These IAPs are characterized by two types of structural motifs: a N-terminal domain containing two or three repeats of 70-80 amino acid repeats termed BIRs (Baculoviruses IAP Repeats) and a C-terminal zinc-binding domain known as RING finger. Studies show that IAPs not only act as inhibitors of specific caspases (Deveraux et al., 1997, Roy et al., 1997), but also, serve as substrates for these proteases (Deveraux et al., 1999). In cytochrome C release-induced apoptosis, XIAP, cl API and cIAP2 can prevent the proteolytic activation of procaspase-9, and subsequent activation of procaspase-3, -6 and -7. These IAPs do not prevent caspase-8-induced activation of procaspase-3, but they can inhibit active caspase-3 directly and block the downstream apoptotic events. Many IAPs are reported to be under the control of NF-kB family of transcription factors. Studies revealed that using viral expression vectors and transgenic mice, downstream mitochondrial permeabilization and caspase activation can be intervened. Therefore, IAPs have emerged as promising therapeutic targets with dual abilities: over-expression to 5 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. make cells resistant to apoptosis in treating neurodegeneration diseases, while inhibition of expression to sensitize cells to cell death in anticancer therapy. Among the caspases, caspase-8 (ICE-Lap5, MACH, FLICE, CAP4) is very well characterized because of its important role as an apical molecule in death receptor signaling pathways. The caspase-8 zymogen possesses significant activity, but its full activity requires proteolytic cleavage of its prodomain (Muzio et al., 1998). Upon the activation of death receptors, the increased local concentration of the caspases results in the auto-proteolytic activation of procaspase-8 (Muzio et al., 1998). Caspase-8 knock-out mice develop prominent abdominal hemorrhage due to hyperemia and manifest impaired formation of cardiac muscles (Varflomeev et al., 1998). Individuals with homozygous caspase-8 mutations not only display characteristic defective lymphocyte apoptosis in autoimmune lymphoproliferative disorder (ALPS), but also exhibit defects in the activation of T, B and natural killer cells, which leads to immunodeficiency. This data suggests a possible dual role of caspase-8 in both negative and positive regulation of immune system in vivo. It also suggests that caspase-8 deficiency in human is compatible with normal development and has a postnatal role in immune activation of na'ive lymphocytes. 1.3 Extrinsic Signaling Pathways in Apoptosis Apoptosis is tightly regulated by interwoven signaling pathways. During apoptosis, uniform execution machinery is triggered by a wide variety of extra- and intracellular signals. As active caspases trigger rapid cell death, there are stringent 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mechanisms to regulate caspases activation. These include synthesizing caspases as zymogens, highly evolved upstream regulatory pathways, and the availability of endogenous inhibitors. Apoptotic signals can be transduced through “intrinsic” pathway mediated by cytochrome C released from mitochrondria into cytosol or through “extrinsic” pathway, where the binding of ligands to the tumor necrosis factor receptor superfamily triggers apoptosis (Peter et al., 1998). These death receptors include Fas (APOI/CD95), TNF-RI, DR3 (Apo3/TRAMP/Wsl-l/LARD), DR4 (TRAIL-RI), and DR5 (TRAIL-2) which all possess a characteristic death domain (DD) within the cytoplasmic region of the receptors (Locksley et al., 2001). 1.3.1. Fas/CD95 Pathway Among these death receptors, Fas (CD95, APO-1) signaling pathway has been extensively studied. Fas was first identified a decade ago using antibodies that induced rapid death of tumor cells (Yonehara et al., 1989; Trauth et al., 1989). Fas is a member of the Tumor Necrosis Factor receptor (TNF) superfamily. Fas is a 319-aa, 45-kDa type I transmembrane protein expressed in various cells and preassembled as trimer (Itoh et al., 1991). As a prototypical member of the tumor necrosis factor receptor (TNFR) superfamily, Fas contains three extracellular cysteine-repeated domains (CRDs) (Naismith et al., 1998) and an 80-aa region of “death domain”, which serves as protein-protein interaction module in cytosolic apoptotic signaling (Tartaglia et al, 1993). Fas ligand (FasL, CD95L, CD178, Apo-1 L) is a type II trimeric transmembrane protein of 40 kDa and can be secreted 7 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. in a soluble form (26 kDa) by the action of metalloproteinases. Soluble and truncated forms of death receptors can block receptor function and those that block TNF action have been used as therapeutic agents in human diseases (Feldman et al., 1998). Like other TNF family members, homotrimeric FasL binds to trimerized Fas (Smith et al., 1994; Grass and Dower, 1995). Aggregation of Fas by Fas Ligand (FasL) or agonistic anti-Fas antibodies results in sequential recruitment of FADD/MORT and procaspase-8 to Fas to form the death-inducing signaling complex (DISC)(Nagata, 1997). The death domain (DD) of FADD binds to DD of Fas through homotypic interactions. Consequently, the death effector domain (DED) of FADD interacts with the DED of procaspase-8 or procaspase-10 (Kischkel et al., 2001). Juxtaposition of procaspase-8 results in autocleavage and the released active caspase-8 activates downstream effector caspases, such as caspase-3 and -7. (Medema et al., 1997; Peter et al., 1998; Salvesen et al., 1999). Bid is activated by cleavage by caspase-8 and subsequently translocates to mitochondria leading to apoptosis through releasing of cytochrome c (Li et al., 1998; Luo et al., 1998). Therefore, the “extrinsic pathway” of Fas-induced apoptosis crosstalks with “intrinsic pathway” through cytochrome C which is linked to Bid. Both naturally occurring and experimentally produced genetic defects that impair DISC formation can severely impair Fas-induced apoptosis. Experimental overexpression of active caspase-8 or induced oligomerization of procaspase-8 is sufficient to cause apoptosis, which reveals that caspase activation is central to Fas signaling (Shuford et al., 1997; Takahashi et al., 1999; Cooper et al., 2002). 8 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Two pathways have been detected in Fas-induced apoptosis (Scaffidi et al., 1998). In type I cells, such as thymocytes in-vitro, Fas-induced apoptosis is refractory to Bcl-2, as sufficient amount of activated caspase-8 cleave and activate caspase-3 and -7. In contrast, in type II cells, such as hepatocytes, apoptotic signaling depends on a mitochondrial step, where caspase-8-cleaved Bid translocates to mitochondria and causes cytochrome C release (Li et al., 1998; Luo et al., 1998; Korsmeyer et al., 2000). The released cytochrome c induces the formation of apoptosome that is composed of seven Afa-1 adapter molecules, one cytochrome c and a dimer of initiator caspase-9 (Liu et al., 1996; Li et al., 1997c; Zou et al., 1997; Cain et al., 2000; and Acehan et al., 2002). Activated caspase-9 can also activate caspase-3 and -7 that execute apoptosis (Figure 1-1 and 1-2). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. c * ll d eath ' i» A > » fip m 'I B e l- 1 liSBvm A A c ttn O ats* f t a i n n Rv <;m m ^ U A U f Pttofr C A P D N A r e p a i r 'V . C ott sshrlnt<»a® MamiMranB feMMMngi DNA tMtpMenhrtkm CNrenwtm cono*rt*jmon ,„ ,,, . . M n . . . ||. .... ~ v APOPTOSIS Figurel-1. Fas/CD95 pathway in apoptosis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Mitochondria! Control of Apoptosis E » rv * » /ia t P. Is ! 'Cytofci c r« i X * * * * A £ ijp tO R S # € A M 7 » iA ^ i 1444 © # d c y 4 * * o * ie f t O Q tM N tt r f t l i e n ■ M U M '* " / | \ ^ * i * slAt* V * S S K . ^ ««xso« ■ » » s / \ l i O M A « i» m » 0 e ' 0«*xafc»*ie Stress & p o m & S I S Figure 1-2. Caspases pathway in apoptosis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The Fas/FasL pathway plays a critical role in three types of physiological apoptosis: 1) peripheral deletion of activated mature T cells at the end of immune response (Nagata et al., 1995); 2) elimination of virus-infected cells or cancer cells by cytotoxic T cells and by natural killer cells; and 3) killing of inflammatory cells at “immune-privileged” sites such as the eye (Griffith et al., 1995). Non-functional Fas-FasL interactions have been identified in several autoimmune diseases (Hayashi et al., 2003). Studies in Fas and FasL mutant mice have extended our understanding of the role these two factors play in the regulation of the immune system. It was first discovered that there are naturally occurring Fas- mutant mice (Ipr mice) which show syndromes of lymphoproliferation mutations (Watanabe-Fukunaga et al., 1992); and FasL-mutant mice (gld mice), which manifest generalized lymphoproliferative disease (Takahashi et al., 1994). It has been reported that mature T cells from Ipr and gld mice do not die after activation. In addition to T cell abnormalities, Fas-deficient mice accumulate B cells and have increased levels of various classes of immunoglobulin (Cohen et al., 1991). Fas transgenic mouse under CD2 promoter has demonstrated the importance of controlling Fas-mediated apoptosis by restoring apoptosis function and ameliorated autoimmune symptoms (Wu et al., 1993). Studies in mouse mutation led to understanding of human diseases. It has been reported that mutations in FasL is associated with human lymphoproliferative syndrome (Rieux-Laucat et al., 1995) and autoimmune lymphoproliferative syndrome (Fisher et al., 1995). Whereas Fas gene mutations has been detected in the Canale-Smith syndrome, an inherited 12 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. lymphoproliferative disorder associated with autoimmunity (Drappa et al., 1996). These patients have an increased risk of developing T- and B- cell lymphomas (Straus et al., 2001). Due to defects in lymphocyte apoptosis, these patients’ risk of non-Hodgkin’s lymphoma and Hodgkin’s disease are 14 and 51 times higher than expected. These findings indicate Fas-mediated apoptosis is important for preventing B-cell and T-cell lymphomas. In Hashimoto's thyroiditis, the infiltration of activated T cells and suicidal FasL-positive thyrocytes causes Fas-mediated apoptosis of thyrocytes. In systemic lupus erythematosus (SLE), the tolerance breakdown of self-antigens leads to the development of autoantibodies. In Rheumatoid arthritis (RA), which is characterized by pronounced hyperplasia of the synovial tissue, cell infiltration and periarticular osteoporosis, the reduced sensitivity to apoptosis in synovial cells and infiltration of autoreactive T and B cells contribute to the formation of the disease. In addition, the essential role of FADD and caspase- 8 in Fas/FasL pathway is revealed in FADD or caspase-8-deficient mice that are resistant to Fas-induced apoptosis. (Roger et al., 2001; Mittler et al., 1999; Sun et al., 2002) A family of viral proteins called vFLIPs (vira-FLICE-like inhibitory protein) is identified from y-herpes viruses and is capable of blocking Fas-induced apoptosis through association with the DISC (Hu et al., 1997; Bertin et al., 1997; Thome et al., 1997). Two cellular homologues, named c-FLIPs (short) and c-FLIPl (long) were identified (Irmler et al., 1997; Srinvasula et al., 1997). c-FLIPl contains two tandem DEDs and an inactive protease-like domain that is homologues to caspase-8. 13 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. c-FL IP l was found to be cleaved in the DISC by caspase-8 (Scaffidi et al, 1999). The role of cFL IP l is controversial, as FLIP either inhibits or activates apoptosis by in vitro or overexpression studies (Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997; Inohara et al., 1997; Irmler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997). In studies using cFILP stably transfected cell line, cFLIP blocks caspase-8 activity at DISC and thereby inhibits Fas-induced apoptosis (Scaffidi et al., 1999). The inhibition of apoptosis is shown to be mediated through recruitment to and cleavage in DISC, resulting in an inactive caspase-8 composed of plO subunit from caspase-8 and plO subunit from cFL IP l. Therefore, the formation of inactive caspase-8 resulting from cFL IP l interference leads to inhibition of apoptosis. It has been shown that IL-2 treatment downregulates c-F L IP l, which is responsible for the Fas-induced apoptosis sensitivity of long-term-activated peripheral mouse T cells (Refaeli et al., 1998). cFLIP may also link Fas signaling to co-stimulation, supported by the results that transfection or transgenic expression of c-FLIP enhanced the activation of both nuclear factor N F - kB and the mitogen-activated protein (MAP) kinase (Kataoka et al., 2000). The activation of these factors led to increased IL-2 secretion. Therefore, in this scheme, caspase inhibitors could act by inhibiting processing of c-FLIP in the Fas signaling complex, which is apparently important for its function. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.3.2 TRAIL Pathway The second major apoptotic signal is triggered by TRAIL/Apo2L (TNF Related Apoptosis Inducing Ligand) (Ashkenazi and Dixitm 1998). TRAIL is a type II 33-35 kDa transmembrane protein that was identified based on its homology with FasL and TNF (Wiley et al., 1995). Its extracellular carboxy-terminal portion can be cleaved in a soluble form and vesicle-associated form (Mariani and Krammer, 1998; Moleon et al., 2001). TRAIL forms homotrimers like other TNF family members (Hymowitz et al., 1999; Mongkolsapaya et al., 1999). Unlike other members of the TNF ligand family, TRAIL carries a zinc ion at the trimer interface coordinated by the single unpaired cysteine residue (Cys 230) of each monomer (Hymowitz et al., 2000). TRAIL is expressed in many tissues, whereas FasL is mainly restricted to activated T cells, NK cells and immune-privileged sites (Wiley et al., 1995). This ligand appears to play an important role in immune surveillance by cells of the innate immune system against viral infection and tumor. It is suggested that TRAIL may play some role in peripheral T cell deletion (Masters et al., 1996). TRAIL has indicated an important role in antitumor surveillance by immune cells in mouse gene knockout studies (Cretney et al., 2002; Smyth et al, 2003). In contrast to TNF, TRAIL has been found to specifically kill tumor cells without harming normal cells. In addition to TRAIL, agonistic antibodies specific for its receptor DR4 (Chuntharapai et al., 2001) and DR5 (Ichikawa et al., 2001) are also being examined for their antitumor activities. It mediates thymocyte apoptosis and is important in the induction of autoimmune diseases (Lamhamedi-Cherradi et al., 2003). 15 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TRAIL induces apoptosis through interacting with its receptors. Tour human homologues have been identified: DR4 (Pan et al., 1997), DR5/KILLER (Pan et al., 1997; Sheridan et al., 1997; Wu et al., 1997), DcRl/TRID/TRAIL-R3 (Degli Esposti et al., 1997a; Pan et al., 1997b; Sheridan et al., 1997), and DcR2/TRAIL-R4 (Degli Esposti et al., 1997; Marsters et al., 1997), as well as a soluble receptor called osteoprotegerin (OPG) (Emery et al., 1998). DR4 and DR5 contain DD in their intracellular region, which is essential for apoptosis signaling. On the other hand, DcRl and DcR2 function as decoy receptors which compete with DR4 and DR5 for binding to TRAIL. The physiological relevance of OPG, which has low affinity for the ligand, remains to be found (Truneh et al., 2000). Ligation of TRAIL to its receptors triggers their trimerization and clustering of their DD, resulting in the formation of DISC (Bodmer et al., 2000; Kischkel et al., 2000; Sprick et al., 2000), which resembles that of Fas. Adapter protein FADD is consequently recruited to DD through homotypic interaction. The DED of FADD in turn interacts with procaspase-8 and -10, resulting in their subsequent activation (Medema et al., 1997). It has been demonstrated that apoptosis induced by TRAIL requires caspase activity (Masters et al., 1996; Mariani et al., 1997; Mariani and Krammer, 1988). However, ectopic expression of FADD-DN in amounts sufficient to block Fas- induced cell death did not block apoptosis triggered by TRAIL. This indicates that a FADD-independent pathway links TRAIL to caspases (Masters et al., 1996). Furthermore, cells from FADD-deficient mice show full responsiveness to DR4; on the contrary, induction by Fas, TNFR1 and DR3 show resistance, which confirms the 16 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. existence of a FADD-independent pathway that couples TRAIL to caspases (Yeh et al., 1998) (Figurel-3). I CARD (Caspase Recruiting Domain) DD (Death Domain) 77 DED (Death ; Effector Domain) j f j > ! Kinase I Domain \ Death Signalling % Pathways \ NF-kB S ignaling Pathways J , Other Signaling Pathways ~ — ~ j Inhibition w RANKL ^ I II to « * ■ ■ • j f l j j ® * ■ * ir FADD.1U, ■ ' » C t» p w * W * to i I O ... M i JN K Mo NF-vB c m l a w Figure 1-3. TRAIL pathway in apoptosis. 17 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The important role played by TRAIL in the regulation of immune system is demonstrated in its ability to inhibit cell-cycle progression in T cells in periphery (Song et al., 2000). Mice that are deficient for TRAIL showed defects in thymocyte apoptosis, such that thymic deletion induced by T-cell receptor is markedly impaired. These mice are also hypersensitive to collagen-induced arthritis and streptozotocin induced diabetes, and develop heightened autoimmune responses (Cretney et al., 2002; Lamhamdi-Cherradi et al., 2003). These results support the notion that TRAIL mediates thymocytes apoptosis. However, TRAIL knockout mice are viable, fertile, and have no obvious haematological defects (Cretney et al., 2002). Mounting evidence has suggested that TRAIL mediates a significant part of the antitumor and antiviral cytotoxicity of dendritic cells, monocytes, NK and T cells. IFN, which regulates TRAIL transcription (Gong and Almasan 2000; Sato et al., 2001), potentiates apoptotic response in fibroblasts infected with human CMV by upregulating TRAIL, sensitizing infected cells (Sedger et al., 1999). The antitumor effects of IFN on multiple myeloma are also mediated through increased expression of TRAIL, which are blocked by dominant negative form of DR5 (Chen et al., 2001). Moreover, IL-2 induces TRAIL expression in human NK cells which kill tumor cells through the ligand (Johnsen et al., 1999). It is shown that TRAIL alone is responsible for the cytotoxicity of measles virus-infected dentritic cells (Vidalain et al., 2000); Furthermore, TRAIL might function to keep inflammatory lymphocytes in check to prevent autoimmune diseases. TRAIL inhibits autoimmune inflammation in experimentally induced rheumatoid arthritis (Song et al., 2000) and 18 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. multiple sclerosis (Hilliard et al., 2001). In addition, TRAIL has been documented in playing a role in immune responses such as organ transplantation (Adams et al., 2002). 1.3.3 TNF Pathway Another major signaling pathway in apoptosis regulation is through TNFR1 receptors. Tumor necrosis factor (TNF) is produced mainly by activated macrophages and T cells in response to infection (Tartaglia and Goeddel, 1992). It plays a pivotal role in orchestrating innate inflammatory responses in vertebrates. TNF triggers local expression of chemokines and cytokines, promoting the adhesion, extravasation, attraction and activation of leukocytes at site of infection. At the later stage of immune response, it facilitates transition from innate to acquired immunity by enhancing antigen presentation and T cell costimulation. To date, 19 different ligands have been identified that belong to the TNF super family. One intriguing feature presented by these ligands is their function as agonists when being cleaved from cell membrane into soluble forms. For instance, membrane-bound FasL eliminates human peripheral-blood T cells; on the contrary, soluble FasL blocks this killing (Suda et al., 1997). The 19 ligands mediate their cellular response through 29 receptors that belong to the TNF receptor (TNFR) superfamily. They are characterized by the presence of cysteine-rich domain (CRD) in the extracellular domain. There are two general groups of TNFR superfamily. One group contains death domain known as death receptors. Death domain is a region containing 19 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. approximately 80 amino acid first identified in the cytoplastic domain of Fas and TNFRs, which lead to abolishment of ligand-induced apoptosis (Itoh et al., 1991; Itoh et al, 1993; Tartaglia et al., 1993). In addition, TNFR1 and TNFR2 contain a conserved extracellular domain that mediates specific ligand-independent assembly of receptor trimers (Chan et al., 2000). This domain is required for the assembly of TNFR complexes but distinct from that contact with the ligand. Therefore, this finding suggests that TNFR and related receptors function as preassociated complex rather than as individual receptor subunits, which changes conformation upon ligand binding. As for the cellular expression patterns of TNF ligand and receptors, almost all of the TNF ligands are expressed by the cells of the immune system, including B cells, T cells, NK cells, monocytes and dendritic cells. However, TNFRs are expressed by a variety of cells. No cell type in the body has yet been found that does not express TNFR1, whereas expression of TNFR2 is mainly by immune cells and endothelial cells. TNFR1 is the primary receptor for proteolytically derived soluble TNF, whereas TNFR2 is the main receptor for membrane-associated TNF. These receptors trigger cascades of signaling pathways, including IkB Kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 or p42/44 mitogen-activated protein kinase (MAPK) cascades, which control gene expression through transcription factors such as NF-kB and AP-1. TNFR1 is a prototypic member of death receptor family involved in both cell survival and apoptosis. The pleiotropic activities of TNFR1 include activation of NF-kB, which leads to induction of a number of antiapoptotic proteins (Shu et al., 20 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1996), and initiation of apoptosis by recruitment of adaptor FADD and caspase-8 (Varfolomeev et al., 1998, Yeh et al., 1998). In triggered apoptosis, the interaction of TNFa with TNFR1 leads to binding of TRADD (Hsu et al., 1995). TRADD then functions as a docking site for several signaling molecules: binding of TRAF2 (Hsu et al., 1996; Rosenwald, 1996) and RIP (Hsu et al., 1996) stimulates activation of N F - kB pathway and JNK/AP1 pathway; whereas the binding of FADD mediates apoptosis cascade (Chinnaiyan et al., 1996; Hsu et al., 1996). The activation of N F - kB and AP-1 leads to proinflammatory and immunomodulatory genes for survival (Tartaglia and Goeddel, 1992). TNF induces a number of antiapoptotic genes, including c-FLIP, cIAPl, cIAP2, A l, A20, TRAF1 and TRAF2. TNF rarely triggers apoptosis unless transcription or protein synthesis is blocked, which implies that apoptosis is suppressed by preexisting cellular factors. Studies revealed that blockage of IK K /N F -kB pathway uncovers TNF’s apoptotic capacity (Chen and Goeddel, 2002; Karin and Lin, 2002). This is supported by studies in mice where TNF- or TNFR- knockout develop normally, however, mice deficient in N F - kB signaling die in utero from TNF-dependent apoptosis of liver cells. It has been suggested that the decision between apoptosis and survival partially depends on the differential DD or DED complexes being formed. Various DD-containing proteins can form distinct complexes in a temporal manner in TNFR1 induced apoptosis (Michaeu and Tschopp, 2003). Complex I is assembled initially within minutes after activation which contains TRADD, TRAF2, cIAPl and the kinase RIP1. Complex I activates IKK and N F - kB survival cascade. In a second step, TRADD-based 21 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. complex I is displaced by complex II, which contains FADD and caspase-8. In this model, the pivot of the balance between death and survival rests on cF L IP l. When sufficient N F - kB is activated, it upregulates cFL IP l. Adequate cF L IP l then inhibits caspase-8 activation in complex II. Therefore, according to this model, apoptosis is induced only when insufficient amount of N F - kB is produced. This two-step model provides explanation about the delay of apoptosis induction comparing to Fas. In addition, from evolution point of view, it gives the cell an opportunity to make the decision between life and death arbitrated by c-F L IP l. This model is also relevant for the possible mechanisms of the inability to recruit FADD and caspase-8 in Type II cell triggered by Fas. As many pathological imbalance between N F - kB regulated survival signals, these two processes could be potential disease treatment (Figure 1-4). situations are characterized by an and caspase-mediated apoptosis targets in cancer and autoimmue R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. C b 'U stuHMIutygo *" ▼ tmm&rmm tMttufngi M A (ragnttmutfcHt ervtomatm c<m«*»wunl©*> , ____________ J St mowxmm Figure 1-4. TNF pathway in apoptosis. The vast majorities of these factors are powerful modulators of critical immune functions and participate in pathogenic mechanism leading to autoimmune disease. The normal function of TNFR1 and TNF is important in maintaining homeostasis of the organism. Germline mutations in the extracellular domain of TNFR1 suppress the secretion of the receptor leading to a family of dominantly inherited auto inflammatory syndromes. (McDermott et al., 1999) 23 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.4 Intrinsic Signaling Pathways in Apoptosis In response to DNA damage, cell cycle checkpoint defects, mitotic catastrophic hypoxia, loss of survival factors or other types of cellular stresses, the intrinsic pathway will be activated. This pathway involves release of cytochrome C from mitochondria (Liu et al., 1996). Then cytochrome C and dATP/ATP act as cofactors in the activation of procaspase 9 by Apaf-1 (Li et al., 1997). Caspase 9 in turn activates downstream caspases, such as caspase-3, -6, and -7 (Faleiro et al., 1997; Li et al., 1997; Muzio et al., 1997). The Bcl-2 family proteins regulate the release of cytochrome c from mitochondria. Bcl-2 family proteins can be divided into two groups according to their ability to either promote apoptosis (Bax, Bak, Bcl-Xs, Bid, Bik, Hrk ...) or suppress it (Bcl-2, Bcl-Xl, Bcl-W, Bh-1, Brag-1) (Newton et al., 1998). The intrinsic and extrinsic pathways cross-link with each other by caspase-8, where it cleaves Bid and promotes cytochrome c release through interaction with Bax and Bak. Activated p53 by DNA damages can transcriptionly activate proapoptotic genes, such as Bax (Miyashita et al., 1994; Zhan et al., 1994; Zhan et al., 1996) and antiapoptotic genes, such as Bcl-XL (Zhan et al., 1996). Also p53 can downregulate Bcl-2 expression (Haidar et al., 1994; Miyashita et al., 1994). In addition, Fas, the ligand of FasL, and DR5, a member of TNFR, can also be upregualted by p53 (Owen-Schaub et al., 1995; Wu et al., 1997). Recently, translocation of p53 following DNA damage to mitochondria has been described (Mihara et al, 2003) and its ability to directly interact with BCL-2 family proteins to regulate cytochrome c release has been noted. 24 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER II A NOVEL MURINE ANTI-APOPTOTIC PROTEIN TOSO PROTECTS FAS-INDUCED APOPTOSIS BY INHIBITING CASPASE-8 ACTIVATION THROUGH ITS BINDING TO FADD 2.1 Abstract Mouse TOSO gene (mTOSO), the homologue of human TOSO gene which inhibits apoptosis mediated by members of the TNF receptor family, was cloned in this study. The cDNA sequences of the TOSO gene in nine strains of normal and disease mice indicated polymorphism or possible missense mutations. The mRNA expression of mTOSO was detected in B cells and T cells, heart, lung, thymus, spleen and small intestine. The subcellular localization of mTOSO protein was detected on the cytoplasmic membrane determined by immunostaining studies. The anti-apoptotic function of cloned mouse TOSO gene was examined in Fas-induced apoptosis in Jurkat cells and was shown to significantly enhance the survival rate. The activity of caspase-8 in Fas induced apoptosis was downregulated in mTOSO transfected cells by enzyme assays measuring its activity of cleaving substrate. In vitro GST-pulldown assay demonstrated that mTOSO binds specifically to FADD molecule, therefore suggesting a mechanism where the binding of TOSO to the FADD complex inhibits the activation of caspase-8, possibly through the disruption of DISC complex formation. Further deletion studies of mTOSO indicated that the C-terminal intracellular domain of TOSO is required for binding to FADD. 25 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The results yielded from these studies suggested that mTOSO inhibits apoptosis by binding to FADD. In the studies of TRAIL-induced apoptosis, mTOSO did not show protection from cell death, which confirms the existence of FADD-independent pathways suggested by other studies. In addition, to assess the anti-apoptotic function of mTOSO in vivo, primary T lymphocytes isolated from mTOSO transgenic mice established by T cell targeted expression showed significant protection from Fas-induced apoptosis, comparing to that of non- transgenic mice. The elevated expression of mTOSO RNA in T lymphocytes in transgenic mice was confirmed by realtime PCR analysis. Because of the important role played by Fas/FasL pathway in autoimmune diseases, results from this study indicates a potential critical role played by mTOSO at the level of whole organism relating to apoptosis and auotoimmune diseases. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.2 Introduction Apoptosis or programmed cell death is a physiology form of cell death that plays a critical role in development and tissue homeostasis in multicellular organisms (Villa et al., 1997; Kidd, 1998). Apoptotic signals can be transduced through “intrinsic” pathway mediated by cytochrome c which is released from mitochrondria into cytosol (Shimizu et al., 1996; Kroemer et al., 1997); or through “extrinsic” pathway, where the binding of ligands to the tumor necrosis factor receptor superfamily triggers apoptosis (Peter et al., 1998). These death receptors include Fas (APOI/CD95), TNF-RI, DR3 (Apo3/TRAMP/Wsl-l/LARD), DR4 (TRAIL-RI), and DR5 (TRAIL-2) which all possess a characteristic death domain (DD) within the cytoplasmic region of the receptors (Locksley et al., 2001). Among these death receptors, Fas signaling pathway has been extensively studied. Fas is a 45 kDa type I transmembrane protein expressed in various cells (Itoh et al., 1991). Aggregation of Fas by Fas Ligand (FasL) or agonistic anti-Fas antibodies results in sequential recruitment of FADD and procaspase-8 to Fas to form the death inducing signaling complex (DISC) (Nagata, 1997; Kischkel et al., 1995). The death domain (DD) of FADD binds to DD of Fas through homotypic interactions. Consequently, the death effector domain (DED) of FADD interacts with the DED of procaspase-8 or procaspase-10 (Kischkel et al., 2001). Juxtaposition of procaspase-8 results in autocleavage and the released active caspase-8 activates downstream effector caspases, leading to apoptosis (Medema et al., 1997; Peter et al., 1998; Salvesen et al., 1999). In TNFa triggered apoptosis, the 27 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. interaction of TNFa with TNF-RI leads to the binding of TRADD (Hsu et a l, 1995). TRADD then functions as a docking site for several signaling molecules: binding of TRAF2 (Hsu et al, 1996; Rosenwald, 1996) and RIP (Hsu et al, 1996) stimulates activation of NF-kB pathway and JNK/AP1 pathway; whereas the binding of FADD mediates apoptosis cascade (Chinnaiyan et al, 1996; Hsu et al., 1996; Varfolomee et al., 1996). DR3 has close sequence similarity to TNFRI and triggers responses resembling those of TNFRI, namely, NF-kB activation and apoptosis (Chinnaiyan et al., 1996; Kitson et al., 1996; Marsters et al., 1996; Bodmer et al., 1997; Screaton et al., 1997). Apoptosis induced by TRAIL, a TNFa homologue, involves ligation of death receptor 4 and/or death receptor 5 followed by recruitment of FADD and caspase-8 into a DISC (Kischkel et al., 2000; Kuang et al., 2000; Sprick et al., 2000). A family of viral proteins called vFLIPs and a cellular homologue cFLIP contain a DED that is similar to the corresponding segment in FADD and caspase-8. The role of cFLIP is controversial, as FLIP either inhibits or activates apoptosis by in vitro or overexpression studies(Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997; Inohara et al., 1997; Irmler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997). In studies using cFILP stably transfected cell line, cFLIP blocks caspase-8 activity at DISC and thereby inhibits Fas-induced apoptosis (Scaffidi et al., 1999). Fas participates in regulation of the immune system for negative selection of T cells, termination of immune responses after antigen stimulation, and cellular immune response (Nossal, 1994; Nagata, 1997; Wallach et al., 1999; Locksley et al., 2001). The importance of regulated Fas-induced apoptosis in immune system has 28 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. been clearly demonstrated by studies in mice with mutated non-functional Fas or FasL. Mice lacking functional Fas (Ipr) (Watanabe-Fukunaga et al., 1992) or FasL (gld) (Takahashi et al., 1994) develop severe autoimmune lymphoproliferative disorders. Human TOSO gene (hTOSO) is identified by cDNA library based functional cloning (Hitoshi et al., 1998). It has been shown that human TOSO gene functions in blocking the Fas-FasL pathway of apoptosis by inhibiting procaspse-8 activation (Hitoshi et al., 1998). hTOSO gene encodes a 390 amino acids protein which belongs to the imunoglobulin gene superfamily. hTOSO mRNA expressions are observed in lymph nodes, spleen, lung and kidney. However, TOSO gene expression is restricted in hematopoietic cell lines. TOSO inhibits Fas-, TNFa- and FADD- induced apoptosis. The expression of TOSO mRNA is up-regulated after T cell activation, suggesting a role of protecting from activation-induced apoptosis in T cells. Expression of TOSO also increased the mRNA level of c-FLIP, indicating a potential molecular mechanism that TOSO's anti-apoptotic function is mediated through c-FLIP molecule. hTOSO gene is located within human chromosome region Iq31-q32, which is associated with various types of hematopoietic malignanceies and solid tumors (Mertens et al., 1997; Mitelman et al., 1997). In addition, studies in nude mice demonstrated that duplication of the chromosome segment of lq l l-q32 is associated with proliferation and metastasis of human chronic lymphocytic leukemic B cells (Ghose et al., 1990). 29 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Since the first report of human TOSO from Hitoshi et al. 1998, no other studies on TOSO has been reported to date. To confirm that TOSO is indeed a functional gene involved in apoptosis, we underwent studies ranging from cloning murine TOSO (mTOSO), conducting similar experiments performed on human TOSO and extending function and mechanism studies of mTOSO. Among the questions to be answered, there are questions such as the putative cell membrane location of TOSO, and the molecular mechanism of how TOSO inhibits caspase-8 activation in Fas-induced apoptosis, etc. In this study, the mouse homologue of human TOSO was cloned, its tissue expression pattern, subcellular localization and its molecular anti-apoptotic mechanism were determined. Mouse TOSO was most abundantly expressed in spleen, and was detectable in heart, thymus and small intestine. Immunostaining assays demonstrated its cytoplasmic membrane subcellular localization. Mouse TOSO inhibited Fas-induced apoptosis in stably transfected Jurkat cells and inhibited caspase-8 activation. In addition, primary T cells isolated from T-cell-targeted mTOSO expression transgenic mice indicated protection from cell death as compared to cells from none-transgenic mice upon FasL induction. Most importantly, our study showed specific binding of cTOSO to FADD through its carboxyl domain by GST pull-down assays. This result indicates that mTOSO can inhibit apoptosis through its binding to FADD, suggesting that the disruption of DISC formation is a possible mechanism of its anti-apoptotic function. Studies of other apoptosis inducers, such as TRAIL, indicated that overexpression of mTOSO 30 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. did not enhance resistance to apoptosis, which confirms the suggestion of FADD independent pathway in TRAIL pathway (Masters et al., 1996; Yeh et al., 1998; Zhang et al., 1998). Given the ample evidence of the importance of maintaining balance between cell proliferation and apoptosis in the immune system, transgenic mice of mTOSO with the targeted expression in T cells was generated to evaluate its importance in autoimmune diseases. The results of these studies will not only extend our knowledge of how TOSO inhibits apoptosis at the molecular level, but also, the understanding of roles played by TOSO in normal immune response and in autoimmunity. Furthermore, the identification of TOSO as a critical control point in apoptotic pathway would function as a rational target for the development in therapeutic and pharmacological treatment in autoimmune diseases. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.3 Material and Methods Materials: Reagents were purchased from the following suppliers: anti-HA mouse monoclonal antibody from Roche; goat anti- mouse and anti-rabbit IgG (H & L) peroxidase conjugated antibodies from Chemicon; Caspase-8 1C 12 monoclonal antibody and FADD antibody (human specific) are from Cell Signaling; polyclonal rabbit anti-Fas (C-20) sc-715 against a C-terminal peptide from Santa Cruz Biotechnology; anti-c-myc mouse monoclonal antibody from Clontech. Cloning the Full Length mTOSO cDNA with the 5' UTR and 3'UTR: BLAST search of human TOSO gene (Flitoshi et al., 1998) against EST-mouse clones was conducted. Clones AA290194, 521993, 509857, and 174968 were purchased (Research Genetics, Inc.) and sequenced. The sequences were analyzed and the putative mouse TOSO (mTOSO) sequence was obtained. Using the primers designed based on the putative mTOSO sequence, Mouse TOSO cDNAs were made from CD3+, CD4+ , CD8+ and CD19+ SW strain mouse cells by RT-PCR (Promega). The 5'-UTR (84 nucleotides) and 3'-UTR (139 nucleotides) were obtained by 5'-RACE and 3'-RACE (Invitrogen), respectively. The sequence of 3'-UTR was determined from two mice strains, NZM2328 and B6, which showed same 3'-UTR sequences. The coding sequence cDNA of mTOSO was cloned into mammalian expression vector pRC/CMV (Invitrogen) at Hindlll/Xbal sites and named pRC/CMV-TOSO. 32 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Plasmid Constructs: All PCR reactions conducted in doing used Pfu high fidelity DNA polymerase enzyme (Stratagen). The plasmid pRC/CMV-TOSO-HA, which encodes HA-tagged mTOSO at the C-terminal was constructed by PCR cloning. Using pRC/CMV-TOSO as the template, TOSO-HA fragment was generated by amplifying with linker primers 5’-CCC AAG CTT TGT GTG TCA GCC TCA CT- 3’ and 5’-GCT CTA GAT CAA GCG TAA TCT GGT ACG TCG TAT GGG TAT TGG CAT GAA GAT CTG GG-3’. The PCR product was digested with Hindlll and Xbal. The PCR fragment containing c-Myc-tag at the C-terminal of mTOSO was amplified by linker primers 5’-CCC AAG CTT TGT GTG TCA GCC TCA CT-3’ and 5’-GCT CTA GAT CAC AGG TCC TCC TCT GAG ATC AGC TTC TGC TCC TCT TGG CAT GAA GAT CTG GG-3’, using pRC/CMV-TOSO as the template. The PCR product was digested with Hindlll and Xbal. The vector pRC/CMV was digested with Hindlll and Xbal. The digested PCR fragments of HA-tagged mTOSO and c-Myc-tagged mTOSO were ligated into the digested pRC/CMV, respectively. All plasmids were propagated in Eschericha Coli DH5a stain (Invitrogen) and extracted by EndoFree Plasmid Maxi Kit (Qiagen). Cell Culture,Transfection: The human embryonic kidney (HEK) cell line 293 and 293T cells were propagated and maintained in DMEM supplemented with 10% FBS. Human T cell lymphoma Jurkat cells were propagated and maintained in RPMI supplemented with 10% FBS. All cells are cultured at 37°C with 5% CO2 in 33 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. humidified atmosphere. Cells were transfected with Effectin reagent (Qiagen) according to manufacture’s instructions. Apoptosis Induction and Measurement: Transiently or stably transfected Jurkat cells, and primary T cells from transgenic mice extracted from spleen were treated with or without various amount of soluble recombinant human Fas Ligand (Alexis) or TRAIL (Alexis) for different time periods at fixed concentration or different concentration for various period as indicated in the figures. Cells were then washed in 1 X PBS, stained by Propidium Iodide (PI) and Annexin V conjugated with FITC in binding buffer (PharMingen), and subjected to FACS analysis. Immunoblotting: Cells were harvested, washed by cold PBS and resuspended in DISC buffer [30 mM Tris-HCl (pH7.5), 1% (w/v) Triton X-100, 150 mM NaCl, l%(w/v) glycerol, 1 mM NasVCL, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), andlO pg/ml of leupeptin and pepstatin. Cells were incubated in DISC buffer for 20 min on ice. Insoluble debris was pelleted by centrifugation at 14,000 X g for 15 min at 4 °C. Following determination of protein concentration by Bio-Rad protein assay (Bio-Rad), samples were denatured by boiling for 5 min in 2XSDS sample buffer [4 M deionized urea, 2% SDS, 5% P-mecaptoethanol, and 62.5 mM Tris-HCl (pH6.8)]. Samples containing 50 pg of proteins were subjected to 8% SDS-PAGE and electro-transferred to PVDF Immobilon-P membrane (Millipore). The membrane was blotted by antibodies described above followed by 34 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. horseradish peroxidase-based chemiluminescence detection using ECL plus western blotting detection reagents (Amersham Biosciences). Caspase-8 Activity Assay: Caspase-8 activities were measured by ApoAlert caspase-8 Assay (Clontech) according to manufacturer’s instructions. Briefly, cells treated with or without FasL for various periods were lysed and the supernatant was mixed with reaction buffer which contained IETD-pNA substrate. Following incubation at 37°C for 2 hours, samples were read at 405 nm by spectrophotometer. Fold-decrease in caspase-8 activity was calculated by substracting the background reading from cell lysates and buffers from the reading of induced and uninduced samples. Northern Blotting: Total RNA from various mouse tissues were extracted using Trizol reagent (Invitrogen), according to the manufacturer’s instructions. 15 pg of total RNA per sample were loaded into RNA agarose gel. RNAs were transferred to positive charged nylon membrane and probed with double stranded full length mTOSO DNA following protocols from Maniatis, 1988. The membrane was T 9 re-probed by p-actin as the loading control. All probes were P labeled using random priming method (Amersham Pharmacia). Immunofluorescence Confocal Microscopy: 293 cells were cultured on overglassed in 6-well plates, transiently transfected with pRC/CMV-TOSO-HA by 35 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Effectin reagent (Qiagen) according to manufacturer’s instructions. 36 hours post-transfection, cells were washed with PBS and then fixed with 3.7% formadehyde for 15 min at room temperature. After washing with wash buffer (1% triton in PBS), specimens were incubated with blocking solution (1% triton, 1% non fat milk and 1% BSA in PBS) for 30 min at room temperature. Then the specimens were incubated with mouse anti-Fas antibody Clone DX2 (Pharmingen) and rabbit anti-HA9 antibodies (Clonetech) for 1 hour at 37°C. After three times 5 min washes with wash buffer at room temperature, secondary antibodies conjugated with goat anti-rabbit IgG conjugated with FITC (Caltag) and rabbit anti-mouse IgG conjugated with Alexa Fluor 568 (Molecular Probes) were then applied and incubated for 1 hour at 37°C. After wash 3 X with PBS, the specimens were subjected to confocal fluorescent microscope. Generation and Genotyping of Ick-TOSO Transgenic Mice: mTOSO cDNA was inserted into the BamI site of the p i017 expression vector containing the lck proximal promoter (Chaffin et al., 1990). The 6.9 kb Notl fragment containing lck proximal promoter, mTOSO, and human growth hormone fragment was then injected into B6DBAF1 blastocysts and implanted into pseudopregnant mice. Founder mice were initially screened by PCR analysis of tail DNA using primers specific for human growth hormone gene hGH (5’-TAG GAA GAA GCC TAT ATC CCA AAG G-3’ and 5’-ACA GTC TCT CAA AGT CAG TGG GG-3’). The transgene-positive lines were further confirmed by Southern blotting analysis probed 36 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. by hGH fragment generated by PCR using the primers listed above. The founder mice were then crossed onto NZM2328 lupus strain background. The distribution of C57/BL-6 and NZM 2328 genetic background in the back crossing mice was determined by 47 micro satellite markers. GST-Pull Down Assays: Plasmid containing wild type and various deletion mTOSO protein were in vitro transcribed and translated in the presence of 3 5 S-methionine (Promega). GST-FADD (gift from Dr. Marcus E. Peter at University of Chicago) was transformed into bacteria and induced by IPTG. GST-FADD fusion protein was immobilized on glutathione-Sepharose beads and incubated with in vitro translated proteins for 2 hrs at 4°C in binding buffer [PBS (1% triton, ImM DTT and ImM PMSF)]. Beads were then washed five times in the binding buffer, and analyzed on SDS-PAGE fluorography. Isolation and Treatment of Primary T cells: Primary T cells from transgenic mice were extracted from spleen and concentrated by passing nylon wool column. Briefly, freshly removed spleen was placed in petridish containing 10 ml complete RPMI-1640 [10% v/v FBS, lOOU/ml penicillin G, 100 pg/ml streptomycin, 1% v/v nonessential amino acids, 2 mM L-glutamine, lOmM HEPES buffer, and 50 pM 2-ME] (Invitrogen). With the plunger of a 1-ml syringe until mesh the tissue. The spleen cells were then washed 2X in complete RPMI, and subject to centrifugation with 1:1 Ficoll/RPMI (Atalanta Biologicals) at 800xg for 25 mins. The middle layer 37 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. containing lymphocytes were extracted and washed 2X in complete RPMI. The cells were then transferred to pre-warmed 200 pm mesh nylon column. Incubate the column 45 min in the upright position in a 37°C, 5% CO2 humidified incubator. T-cells were then eluted in the first 15 ml fraction with warm complete RPMI. Primary T cells were then treated with or without various amount of soluble recombinant human Fas Ligand (Alexis) for different time periods. Cells were then washed in 1 X PBS, stained by Propidium Iodide (PI) and Annexin V conjugated with FITC in binding buffer (PharMingen), and subjected to FACS analysis. Real-Time PCR of Transgenic mTOSO Expression: Spleen from mTOSO transgenic mice, B6 mice and NEM2328 mice were removed and primary T cells were extracted as described in “Isolation and Treatment of Primary T cells”. Total RNA was isolated following manufacturer’s instruction (Trizol, Invitrogen) and cDNA was made using Taqman Gold RT-PCR Kit (Applied Biosystems) according to manufacturer’s protocol. The Taqman PCR was performed separately for TOSO primers and (3-actin primers designed by software provided by the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Primers for mTOSO: toso-lS: 5’-CAC CAG ACT TCA TGA GCA AAG G-3’; toso-2AS: 5’-CCC TCG GTC TTC TCT CCC ATA-3’ (Integrated DNA). Probe for mTOSO: 5’ 6FAMd(CACGCCACCATGGCCCACA)BHQ-1 -3’ (Biosearch Technologies). Primers for p-actin: actin-S: 5’-CCC ACA TAG GAG TCC TTC TAG CC-3’; actin AS: 5’-GAG CAC AGC TTC TTT GCA GCT - 3 ’ (Integrated DNA). Probe for p 38 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. actin, actin-probe: 5’HEX-d(TTGCCGGTCCACACCCGCCACCAGT)BHQ-2-3’ (Biosearch Technologies). Thermal cycles for RT-PCR: 25 °C for 10 min, 48°C for 30 min, 95 °C for 5 min, 4 °C forever. Thermal cycles for PCR: step 1: 50 °C for 2 min; step 2, 95 °C for 10 min; step 3, 95 °C 15 second; step 4, 60 °C 1 min; step 5, go to step 3 for 40X; step 6, end. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.4 Results Identification and Sequence Analysis of Mouse TOSO Using the sequence of human TOSO gene (Hitoshi et ah, 1998), which blocks Fas mediated apoptosis, the EST data bases were searched by saturated BLAST for the mouse clones which contains human TOSO homologue. This search identified four mouse EST clones: AA290194, 521993, 509857, and 174968. These clones were purchased (Research Genetics), sequenced and hypothetical open reading frame (ORF) was identified. The homology of human TOSO and mouse TOSO on DNA level and protein level were compared as shown in figure 1A and figure IB, respectively. The mouse TOSO CDNA shares 70.2% homology (Fig. 2-1A) and protein has 57.7% homology (Fig. 2-1B) with those of human TOSO which contains 390 amino acids. This ORF encodes a 422-amino acid protein, which was named mouse TOSO. The cDNA sequence was confirmed by amplifying mTOSO cDNA from CD3+, CD4+, CD8+ and CD19+ B6 strain mouse cells. The predicted ORF is initiated by ATG start codon within a Kozak context. The cloned cDNA insert of mTOSO contains a 84 nucleotide 5’-UTR, a 1269-nucleotide coding region and a 139-nucleotide 3’-UTR. Analysis of Kyte-Doolittle hydropathy plot displayed striking similarity of mouse TOSO to human TOSO (Fig. 2-2). Computer analysis of mTOSO protein indicated a leader sequence corresponding to amino acid 1 to 17 and a transmembrane domain spreading from amino acid 258 to 286. It was also predicated by the computer analysis that mTOSO is a type I membrane protein with intracellular C-terminal domain. 40 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. cDNA Alignment of Human TOSO with Mouse TOSO hTO SO ATGGACTTCTGGCTTTGGCCACTTTACTTCCTGCCAGTATCGGGGGCCCTGAGGATCCTC mTOSO ATGGACTTTTGGCTTTGGTTACTTTACTTCCTGCCAGTGTCTGGGGCTCTGAGAGTCCTC kk kk kk kk kk kk kk kk k kkk kkkkkkkkkkkkkkk k k k k k k k k k k k k k k k k k hTOSO CCAGAAGTAAAGGTAGAGGGGGAGCTGGGCGGATCAGTTACCATCAAGTGCCCACTTCCT mTOSO CCAGAAGTACAGCTGAATGTAGAGTGGGGTGGATCCATTATCATCGAATGCCCACTCCCT k'k'kk'k'k'k'k-k k k k k k * * * kkk k k k k k kkk k k k k k kkkk kk kk k k k hTO SO GAAATGCATGTGAGGATATATCTGTGCCGGGAGATGGCTGGATCTGGAACATGTGGTACC mTOSO CAACTACACGTAAGGATGTATCTGTGTCGGCAGATGGCCAAACCTGGGATATGCTCCACT * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTO SO GTGGTATCCACCACCAACTTCATCAAGGCAGAATACAAGGGCCGAGTTACTCTGAAGCAA mTOSO GTGGTGTCCAACACC TTTGTCAAGAAGGAATATGAAAGGCGAGTCACCCTGACGCCA * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTOSO TAC C CAC GCAAGAAT CTGTTCCTAGTGGAG G TAACACAG C T GACAGAAAG TGACAGCGGA mTOSO TGCTTGGATAAGAAGCTATTCCTAGTGGAGATGACACAGCTGACGGAAAATGACGATGGA * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTOSO GTCTATGCCTGCGGAGCGGGCATGAACACAGACCGGGGAAAGACCCAGAAAGTCACCCTG mTOSO ATCTATGCCTGTGGTGTGGGCATGAAGACAGACAAAGGCAAGACCCAGAAAATCACCCTG kkkk kkkkkk k k k kk kk kk kk k k k k k k k k k kkkkkkk kkk kk kkkk kk kk hTO SO AAT GTCCACAGT GAATAC------- GAGCCATCATGGGAAGAGCAGCCAATGCCTGAGACTCCA mTOSO AATGTCCATAATGAATACCCAGAACCATTCTGGGAAGATGAATGGACCTCTGAGCGGCCA k k kk kk kk k k k k k k k k k k k k k k kk kk k k k k k k k k k k k k k hTOSO AAATGGTTTCA------------TCTGC---------CCTATTT-----------GTTCCAGATGCCTGCATATGCC mTOSO AGATGGTTGCACAGATTTCTGCAGCACCAGATGCCCTGGCTCCACGGGAGTGAACATCCC k k k k k k k k k k k k k k k k k k k k k k k k k k k k k k hTO SO AGTTCTTCCAAATTCGTAACCAGAGTTACCACACCAGCTCAAAGGGGCAAGGTCCCTCCA mTOSO AGCTCTTCTGGAGTCATAGCCAAAGTTACCACGCCAGCTCCAAAGACTGAGGCCCCTCCG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * k k k k k k k k k hTO SO GTTCACCACTCCTCCCCCACCACCCAAATCACCCACCGCCCTCGAGTGTCCAGAGCATCT mTOSO GTTCACCAGCCCTCCAGCATCACTT CAG TAAC C CAACAT C C CAGAG T T TACAGAG C AT T T kk kk kk kk k k k k k kk kkk k k k k k k k k kk kkk k k kk kkkkkk k hTO SO TCAGTAGCAGGTGACAAGCCCCGAACCTTCCTGCCATCCACTACAGCCTCAAAAATCTCA mTOSO TCTGTGTCAGCTACCAAGTCCCCAGCGCTCCTGCCAGCAACCACAGCCTCAAAGACTTCC kk kk kkk k k kkk kkk k k kk kk kk kk k kk kkk kkkkkkkk k kk Figure 2-1 A. Alignment of cDNA sequences of human TOSO and mouse TOSO. The mouse TOSO DNA sequence has 70.2% homology with human TOSO cDNA. Symble * denotes same necleotide, symble - denotes gap 41 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. hTO SO GCTCTGGAGGGGCTGCTCAAGCCCCAGACGCCCAGCTACAACCACCACACCAGGCTGCAC mTOSO ACTCAGCAAG CAATCAGGCCCCTAGAGGCCAGCTACAGCCACCACACCAGACTTCAT kkk k k k •kkk kkkkk k kk kk kk kk k kkkkkkk kkk kk kk kk hTO SO AG G CAGAGAG CACTGGACTATGGCTCACAGTCTGGGAGG GAAG G C CAAG G----------------------- mTOSO GAGCAAAGGACACGCCACCATGGCCCACACTATGGGAGAGAAGACCGAGGGCTTCACATC * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTO SO ------------------------ ATTTCACATCCTGATCCCGACCATCCTGGGCCTTTTCCTGCTGGCACTT mTOSO CCCATCCCAGAATTTCACATCCTGATTCCGACCTTCCTGGGCTTTCTCTTGCTGGTTCTT kk kkkkkkkkkkkkk k k k k k k kk kk kk kk k k k k k k k k k k kkk hTOSO CTGGGGCTGGTGGTGAAAAGGGCCGTTGAAAGGAGGAAAGCCCTCTCCAGGCGGGCCCGC mTOSO TTGGGACTGGTGGTAAAAAGAGCCATTCAAAGGAGGAGAGCCTCCTCCAGACGTGCGGGC kkk k kk kk kk kk k k k k k kkk k k kk kk kk kkk kkk k k k k k k k k k k k k k hTOSO CGACTGGCCGTGAGGATGCGCGCCCTGGAGAGCTCCCAGAGG— CCCCGC------------------- GG mTOSO CGACTGGCGATGAGGAGGCGAGGCCGGGGGGCTTCCCGCCCGTTCCCCACACAGCGCTGG kk kk kk kk k k k k k k kkk k k k k k k kkkk k kkk k k hTO SO GT--------- CGCCGCGACCGCGCTCCCAAAACAACATCTACAGCGCCTGCCCGCGGCGCGCT mTOSO GATGCCCCGCAGAGGCCGCGCTCGCAGAACAACGTCTACAGCGCCTGCCCCCGGCGCGCA k kkk k k kk kk kk kk k k k k k k k k kkkkkkkk kkk kk kkk kkkk kk kk hTOSO CGTGGAGCGGACGCTGCAGGCACAGGGGAAGCCCCCGTTCCCGGCCCCGGAGCGCCGTTG mTOSO CGGGGACCAGACAGCTTGGGTCCAGCGGAGGCTCCGCTCCTCAACGCCCCAGCCTCAGCG * * * * * * * * * k k kkk kkk k k k k k k k k k k kkk k hTOSO CCCCCCGCCCCGCTGCAGGTGTCTGAATCTCCCTGGCTCCATGCCCCATCTCTGAAGACC mTOSO TCCCCCGCTTCTCCGCAGGTACTTGAAGCTCCTTGGCCCCACACCCCATCTCTGAAGATG * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTO SO AGCTGTGAATACGTGAGCCTCTACCACCAGCCTGCCGCCATGATGGAGGACAGTGATTCA mTOSO AGCTGTGAATACGTGAGCTTGGGCTACCAGCCTGCTGTCAACCTGGAAGACCCTGATTCA kkkkkkk kkk kkk kkk kk k k kk kk kk kkk k k k k kkk k k k k k k k hTO SO GATGACTACAT CAAT GTTCCTGCC mTOSO GAT GAT TACAT CAATAT T CCT GAC Figure 2-1 A. Alignment of cDNA sequences of human TOSO and mouse TOSO. The mouse TOSO DNA sequence has 70.2% homology with human TOSO cDNA. Symble * denotes same necleotide, symble - denotes gap. 42 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Protein Alignment of Human TOSO with Mouse TOSO hTO SO M DFW LW PLYFLPVSGALRILPEVKVEGELGGSVTIKCPLPEMHVRIYLCREMAGSGTCGT mTOSO M DFW LW LLYFLPVSGALRVLPEVQLNVEW GGSIIIECPLPQLHVRMYLCRQMAKPGICST k k k k k k kkkkkkk kk kk kkkk k kkk k kkkk kkk kkk k k k k k k hTO SO VVSTTNFIKAEYKGRVTLKQYPRKNLFLVEVTQLTESDSGVYACGAGMNTDRGKTQKVTL mTOSO VVSNT-FVKKEYERRVTLTPCLDKKLFLVEMTQLTENDDGIYACGVGMKTDKGKTQKITL hTO SO NVHSEY-EPSW EEQPM PETPKW FHLPYLFQM P---------- AYAS SSK FVTRVTT PAQRGKVP P mTOSO NVHNEYPEPFWEDEWTSERPRWLHRFLQHQMPWLHGSEHPSSSGVIAKVTTPAPKTEAPP hTO SO V HH SSPTTQ ITH RPRV SRA SSV A G D K PRTFLPSTTA SK ISA LEG LLK PQ TPSY N H H TRLH mTOSO V H Q PSSITSV TQ H PR V Y RA FSV SA TK SPA LLPA TTA SK TSTQ Q A I-R PLEA SY SH H TR LH hTO SO RQRALDYGSQSGREGQG--------------- F H I L I PTILGLFLLALLGLVVKRAVERRKALSRRAR mTOSO EQ RTRH H G PH Y G RED RG LH IPIPEFH ILIPTFLG FLLLV LLG LV V K R A IQ RR R A SSR R A G k k k kkk k k k k k k k k k k k k kk kk kk kkk k k k kkk k hTO SO------- RLAVRM RALES S ----------QRPRGSPRPRSQNNIYSACPRRARGADAAGTGEAPVPGPGAPL mTOSO RLAMRRRGRGASRPFPTQRWDAPQRPRSQNNVYSACPRRARGPDSLGPAEAPLLNAPASA * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hTOSO PPAPLQVSESPWLHAPSLKTSCEYVSLYHQPAAMMEDSDSDDYINVP mTOSO SPASPQVLEAPW PHTPSLKM SCEYVSLGYQPAVNLEDPDSDDYINIP Figure 2-IB. Alignment of amino acid sequences of human TOSO and mouse TOSO gene products. The mouse TOSO protein has 57.7% homology with human TOSO protein. Symble * denotes same amino acid, symble - denotes gap. 43 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. -3- - 4 - I i I i I i I i I i I i I i I i I i I < I i I i I i I i I i I i I i ! (I 21) 40 60 80 100 120 140 180 ISO 200 220 240 280 280 300 320 340 i I i I i I i I , I i I . I i I i I i I , I , I i I i I i I , I , I i I , I M 80 100 120 140 160 180 200 220 240 260 200 300 320 340 360 300 400 420 Figure 2-2. Kyte-Doolittle hydropathy plot analysis of human TOSO (390 a.a.) and mouse TOSO (422) proteins. Both proteins process a hydrophobic leader sequence and a transmembrane domain as shown in the plot. The overlapping pattern of these two proteins indicates strong similarity of their hydrophobicity. The predicated molecular weight of mouse is about 47 Kda. The cytoplasmic region has a basic amino acid-rich region and an acidic amino acid-rich region. The extracellular domain has homology to the immunoglobulin variable region (IgV) domains. The cytoplasmic region of has partial homology to FAST kinase. The cDNAs have been sequenced inNZM2328, BalB/C, SJL, NOD, B6, C3H, NZW, and 129/SV strains. Sequence variations have been found in these different strains. Whether these polymorphrisms are functionally important or not remains to be determined (Fig 2-3). 44 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. SJL NZW NZM2328 NZB HDD C3H Ba Ib /c BB 129/SV SJL NZU NZM2328 NZB NQD C3H Ba Ib /c BB 129/SV S JL NZU NZN2328 NZB NOB C3H Ba L b /c BB 1 2 9 /S V 15 15 15 15 15 15 15 15 15 20 20 2i 20 20 20 20 20 20 30 30 30 30 30 30 30 30 30 1B0 G3EHPS GSEHPS GSEHPS GSEHPS GSEHPS GSEHPS GSE GSE GSE 210 9 S jJSs I I £ a f* 1 £ SjfE | £ |jjfe J S S jlffE! ? 170 IFlKVTT I flKVTT I flKVTT I flKVTT I flKVTT I flKVTT I flKVTT I flKVTT I flKVTT 220 180 190 200 310 MRFiRGRGflS MRRRGRGflS HRRRGRGflS NRRRGRGHS HRRRGRGflS MRRRGRGflS MRRRGRGflS MRRRGRGflS 1 g k g 1 g ! g !g ! g i K ! g ■ 230 QQflIR PLEfiS 3QHIR PLEflS riQfl I RPLEflS jO flIR P L E flS Q Q flIR PLEflS QOfiIRPLEfiS 3Q flIR P LE flS M flIR P L E flS QOfiIRPLEflS 330 240 v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ v'SHHTRLHEQ 340 250 ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG ITRHHGPHVG 350 'DSLGPfiEfiP D SLGPflEflP 'DSLGPflEflP ’DSLGPfiEfiP 'DSLGPflEflP D SLGPflEflP ■DSLGPflEflP DSLGPSEflP DSLGPNEflP 200 280 200 200 200 200 200 200 280 250 250 250 250 250 250 250 250 250 350 350 350 350 350 350 350 350 350 Figure 2-3. The partial alignment of mTOSO amino acid sequences in normal (B6, C3H, 129/SV, NZW, Balb/c and SJL) and autoimmune (NOD, NZM2328) mouse strains. Identical sequences among all strains were not shown. Mouse TOSO mRNA is Highly Expressed in Adult Spleen To examine the tissue expression pattern of TOSO gene, mRNAs from various tissues of adult B6 mice were extracted. Probed by the coding sequence of mouse TOSO, Northern Blot assay demonstrated strong expression in spleen with a major transcript of 2.3 Kb, along with two minor larger transcripts of 3.8 Kb and 5.8 Kb expressed at lower abundance (Fig. 4). Similarly, human TOSO transcripts in spleen also showed three larger transcripts (2.8, 3.5, and 4.3 kb) in spleen and lymph 45 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. nodes (Hitoshi et al., 1998). These larger transcripts might be the result of alternative splicing in these tissues or the products of incomplete processed messages. It could also be the results of non-specific binding of mTOSO with other spleen specific genes. However, the expression of mTOSO was not detected in kidney or liver of mice as in its human counterparts. Low level expression of 2.3Kb transcript was also detected in heart, lung, thymus and small intestine, without any detection of the other two larger transcripts (Fig. 2-4). No expression was detected in brain, liver, kidney, muscle, testis, or uterus (Fig. 2-4). The mRNA expressions of mTOSO were also observed in B cells and T cells in B6 mice by RT-PCR assays. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. T issu e E x p re ssio n o f M o u s e T O S O m R N A mTOSO ^_p-Actin Figure 2-4. Tissue expression of mouse TOSO. Northern Blot analysis of total RNA (15 pg per lane) from various tissues of B6 mice strain, using cDNA probes corresponding to full-length coding region of mouse TOSO and p-actin. 47 r Further reproduction prohibited without permission. Reproduced w*h permission o, the copyright owner. Further Mouse TOSO Is a Cytoplamic Membrane Protein To prove the subcellular localization predicted by computer analysis, confocal microscopy was performed by immunostaining HA-tagged TOSO in HEK 293 cells. 293 cells were transiently transfected with pRC/CMV-TOSO-HA, which encodes C-terminal tagged mouse TOSO driven by CMV promoter. 36 hours post transfection, mouse TOSO was detected by rabbit anti-HA antibody and stained with anti-rabbit IgG conjugated with FITC (green) in the permealized cells by immunostaining (Fig.2-5 left). As control, the cell surface marker Fas was recognized by mouse anti-human Fas antibody clone DX2 and stained with anti-mouse IgG conjugated with Alexa Fluor 568 (red) (Fig.2-5 middle). The overlapping of Fas and TOSO localizations was shown in the merged image (Fig. 2-5 right). This result confirmed the predicated cell surface localization of mouse TOSO. Furthermore, the super-imposed images of murine TOSO and human Fas strongly suggest the co-localization of these two molecules. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. FAS and mTOSO-HA Staining in 293 FAS mTOSO-HA Superimposed Figure 2-5. Mouse TOSO protein locates on cell membrane. Analysis of HA-tagged mouse TOSO at C-terminal was performed by transiently transfecting HEK 293 cells with pRC/CMV-mTOSO-HA. 36 hr post-transfection, cells were stained with mouse anti-Fas antibody (left, red) and rabbit anti-HA antibody (right, green). Mouse TOSO Protects Jurkat Cells from Fas-induced Apoptosis To assess the antiapoptotic function of cloned mTOSO in Fas/FasL pathway, plasmid pRC/CMV-mTOSO, which contains the full-length mTOSO cDNA and a neo G418 resistant cassette, was stably transfected to T-lymphocyte Jurkat cell line. As the negative control, cells were transfected with vector pRC/CMV. To determine the mRNA expression of transfected mouse TOSO gene, RT-PCR was performed. As shown in figure 2-6A, three positive clones (clones TF-12, TH7 and TB9) express mTOSO mRNA, whereas parental Jurkat cells, three negative clones (clones TC6, 49 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. BC7, and TH4) that were transfected with the vector only do not express the transfected gene. To test whether ectopic expression of mouse TOSO could protect cells from apoptosis, and parental Jurkat cells and stably transfected clones were treated with various concentrations of Fas Ligand (FasL) (0-20 ng/ml) for 16 hours to induce apoptosis. As shown in figure 2-6B, clones with expression of mTOSO significantly protected cells from apoptosis as compared to Jurkat cells and cells with vector only. This result clearly demonstrated that mouse homologue of human TOSO was successfully cloned and that the ectopic expression of mouse TOSO could protect cells from Fas-induced apoptosis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. RT-PCR of mTOSO Gene in Stably Transfected Clones TB-9 TH-4 BC-7 TH-7 Ju rk a t TF-12 TC-6 < J > P lasm id Figure 2-6A. Jurkat cells were stably transfected with plasmid pRC/CMV-TOSO which contains mTOSO gene under CMV promoter. Positive and negative clones were screened by RT-PCR with primers specific to mouse TOSO gene. The PCR product of 0.44 Kb indicates the mRNA expression in the clones. Clones TB-9, TH-7, and TF-12 express mTOSO, whereas clones TH-4, BC-7, TC-6 and Jurkat cells do not express the gene. < D denotes negative control without cDNA template. Plasmid pRC/CMV-mTOSO used as positive control. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Survival rate of TOSO transfected clones 0s JS -M < u Q 1.0 0.8 0.6 TH 7 (TO SO +) TB9 (TO SO +) TF12 (TO SO +) TC6 (TOSO -) BC7 (TOSO -) TH4 (TOSO -) JK1 (TOSO -) 0.4 0.2 0.0 15 0 5 10 20 FasL concentration (ng/ml) Figure 2-6B. Ectopic expression of mouse TOSO protects Jurkat cells from FasL induced apoptosis. Jurkat cells with stably transfected mTOSO (clones TF-12, TH7 and TB9), Jurkat cells with stably transfected vector (clones TC6, BC7, and TH4), and parental Jurkat cells were treated with various amount of FasL for 16 hrs. The survival rates were determined by FACS by staining cells with Annexin V and Propidium Iodide. 52 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Mouse TOSO Protects Jurkat Cells from TNFa-induced Apoptosis Since the Fas receptor has homology to the TNFa receptor R l, and these two receptors share analogous signaling systems as well as several intracellular mediators (Hus et ah, 1996), we examined whether mouse TOSO can protect Jurkat cells from TNFa-induced apoptosis. Jurkat cells and mouse TOSO stably transfected Jurkat cell clone TF12 were treated with 900ng/ml TNFa in the presence of 0.1 pg/ml cycloheximide for 12 hours to induce apoptosis. As shown in figure 2-7, TOSO protected cell from TNFa-induced apoptosis in comparision to Jurkat cells. This result is in agreement with the founding from Hitoshi et al.. (1998). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TNFa Treatment of Mouse TOSO Transfected Jurkat Cells 60% n □ TNF(-) ■ TNF(+) Jurkat (TOSO-) TF12 (TOSO+) Figure 2-7. Mouse TOSO protects Jurkat cells from TNFa-induced apoptosis. Jurkat cells and mouse TOSO stably transfeced Jurkat cell clone TF12 were treated with treated with 900ng/ml TNFa in the presence of O.lpg/ml cycloheximide for 12 hours to induce apoptosis. The survival rates were determined by FACS by staining cells with Annexin V and Propidium Iodide. Mouse TOSO Inhibits Caspase-8 Activation in Fas-induced Apoptosis The extrinsic apoptotic signal triggered by binding of Fas ligand to Fas is transduced through the sequential assembly of Death-Inducing Signaling Complex (DISC), which is composed of adaptor protein FADD and procaspase-8 (Nagata, 1997); therefore, the activity of the apical molecule caspase-8 in this pathway was examined. mTOSO stably transfected clone TF-12, along with the control clone BC-7 cells, were induced for apoptosis by treating with FasL for different periods of 54 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. time at 50 ng/ml concentration. The activation of caspase-8 was determined by enzyme assays measuring its protease activity capable of cleaving substrate IETD- pNA. As shown in figure 2-8, the expression of mTOSO significantly inhibited the activity of caspase-8, comparing to control cells transfected with vector only. This result was consistent with the study of human TOSO, which inhibited pro-caspase-8 activation by Western Blot assays (Hitoshi et al., 1998). Time Course of Caspase-8 Activity in Jurkat Cells transfected with or without mTOSO □ BC-7 (Jurkat) ■ TF-12 (TOSO) • S 1 0 1hr 1.5 hr 3 hr 6 hr 16hr FasL (50 ng/ml) Treatment Figure 2-8. Mouse TOSO inhibits caspase-8 activation in Fas-induced apoptosis. Jurkat cells stably transfected with vector pRC/CMV (clone BC-7) and with mTOSO (clone TF-12) were treated with 50 ng/ml FasL for various times. The caspase-8 activity was measured by its protease activity capable of cleaving substrate IETD pNA. 55 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Mouse TOSO Binds Specifically to FADD through Its C-terminal Domain The activation of pro-caspase 8 is attained by being recruited to FADD molecular (Medema et al., 1997) and subsequent formation of DISC complex (Nagata, 1997; Kischkel et al., 1995). Therefore, the disruption of FADD and caspase-8 binding by mTOSO could be one of the potential mechanisms of how mTOSO execute its anti-apoptotic function. To test this hypothesis, in vitro protein binding assays were performed. As demonstrated in figure 2-8, 3 5S-methionine labeled in vitro translated full-length mTOSO protein bound to immobilized GST-FADD protein (lane 8), whereas it did not bind to GST (lane 5). In addition, the addition of unlabled mTOSO competed with the binding of 35S labeled mTOSO, which indicated the binding specificity between mTOSO and FADD molecules (Figure 2-9, lanes 9 and 10). To further determine the specific domain in mTOSO involved in binding, various deletion mutations of mTOSO were also studied in the GST-FADD pull-down assays. Construct #1 represents full-length protein (1-422), construct #2 (a.a. 16-422), construct #3 (a.a.232-422), construct #4 (a.a.291-422), construct #5 (a.al-290), and construct #6 (a.a. 1-231) are illustrated in figure 2-10A. The results indicated that the cytoplasmic C-terminal domain of mTOSO was required for binding, but not the extracellular N-terminal domain (Figure 2-10B, C, and D). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mTOSO Interacts with FADD GST-FADD Input O (O O a > ( 0 re a o 3 e 3 2 1 o r Figure 2-9. mTOSO specifically binds to FADD. S-methionine labeled in vitro translated full-length mTOSO (lanes 3, 5, 8, 9 and 10), luciferse (lanes 2 and 7) and in vitro translation without DNA template (denotes O, lanes 1, 4, and 6) were incubated with GST-FADD and GST, respectively. TOSO specifically binds to FADD (lane 8), but not to GST (lane 5). Competition between cold and 3 5 S-labeled mTOSO in binding to GST-FADD (lane 10) were also performed. 57 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 10A //- i C onstruct #1 i C onstruct #2 C onstruct #3 C onstruct #4 C onstruct #5 1 ; 1 C onstruct #6 i 10B TOSO Deletions I ----------------------------------1 Luc < J > 1 2 3 4 5 6 Figure 2-10A and 2-10B. Schemata of mouse TOSO deletions in pRC/CMV Vector. In figure 9A, Construct #1 represents full-length protein (1-442), construct #2 (a.a.16-422), construct #3 (a.a.232-422), construct #4 (a.a.291-422), construct #5 (a.a. 1-290), and construct #6 (a.a. 1-231). mTOSO of various deletions were in vitro translated in the presence of 3 5S-methionine and analyzed on 10% SDS-PAGE. Translation without DNA template (denotes < E > ) and Luciferase were used as negative control in the experiment (denotes Luc). 58 mTOSO -M pRC/CMV 16 258 286 422 16 422 232 422 291 422 290 231 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 10C GST Luc O 10 D GST - FADD Luc < £ 1 Figure 2-10C and 2-10D. C-terminal of mTOSO binds to FADD. mTOSO of various deletions were in vitro translated in the presence of 35S-methionine and incubated with immobilized GST-FADD and GST, respectively. #1 represents full length protein (1-442), #2 (a.a.16-422), #3(a.a.232-422), #4(a.a.291-422), #5 (a.a 1-290), and #6 (a.a. 1-231). 59 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Mouse TOSO Does Not Protect Jurkat Cells from TRAIL Induced Apoptosis TRAIL/Apo2L is a member of TNF superfamily that induces apoptosis through engagement of death receptors. TRAIL is a type II transmembrane protein that was identified and cloned based on its sequence homology with TNF and CD95L (Wiley et al., 1995; Pitti et al., 1996). TRAIL induces apoptosis through interacting with its receptors. Four human homologues have been identified: DR4 (Pan et al., 1997), DR5/KILLER (Pan et al., 1997; Sheridan et al., 1997; Wu et al., 1997), DcR 1 /TRID/TRAIL-R3 (Degli-Esposti et al., 1997; Pan et al., 1997b; Sheridan et al., 1997), and DcR2/TRAIL-R4 (Degli-Esposti et al., 1997; Marsters et al., 1997), as well as a soluble receptor called osteoprotegerin (OPG) (Emery et al., 1998). DR4 and DR5 contain DD in their intracellular region, which is essential for apoptosis signaling. On the other hand, DcRl and DcR2 function as decoy receptors which compete with DR4 and DR5 for binding to TRAIL. The physiological relevance of OPG, which has low affinity for the ligand, remains to be found (Truneh et al., 2000). Ligation of TRAIL to its receptors triggers their trimerization and clustering of their DD, resulting in the formation of DISC (Bodmer et al., 2000; Kischkel et al., 2000; Sprick et al., 2000), which resembles that of Fas. Adapter protein FADD is consequently recruited to DD through homotypic interaction. The DED of FADD in turn interacts with procaspase-8 and -10, which results their subsequent activation (Medema et al., 1997; Sprick et al., 2002). It is of interest to determine whether mouse TOSO could enhance the resistance to apoptosis triggered by TRAIL. To examine the function of mTOSO in TRAIL induced apoptosis, Jurkat 60 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. cells stably transfected with mTOSO (clone TF-12), the parental Jurkat cells, and control Jurkat cells transfected with vector only (clone BC-7) were treated with various amount of TRAIL for 16 hrs and the cell survival rates were determined by FACS as described in Material and Methods. The results indicated that over-expression of mTOSO does not significantly protect cells from TRAIL induced apoptosis, comparing to the controls (Figure 2-11). Apparently, this result is against the expected protection function by mTOSO because of the involvement of FADD and caspases in the currently known TRAIL pathway (Masters et al., 1996; Mariani et al., 1997; Mariani and Krammer, 1988). However, ectopic expression of FADD-DN in amounts sufficient to block Fas induced cell death did not block apoptosis triggered by TRAIL. This indicates that a FADD-independent pathway links TRAIL to caspases (Masters et al., 1996). Furthermore, cells from FADD-deficient mice show full responsiveness to DR4, on the contrary, induction by Fas, TNFR1 and DR3 show resistance, which confirming the existence of a FADD-independent pathway that couples TRAIL to caspases (Yeh et al., 1998; Zhang et al., 1998). Therefore, this result is in support of the notion that multiple pathways involving other yet to be determined factors exist in TRAIL triggered apoptosis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TRAIL Treatment of mTOSO Transfected Jurkat Cells 100% BC (TOSO-) TF(TOSO+) Jurkat 90% 80% O ) < c c f f l o l- 70% 60% 0 ) 50% 40% a 30% O 20% 1 0 % 250 300 350 50 100 150 200 0 TRAIL Treatm ent (ng/ml) Figure 2-11. mTOSO does not protect cells from TRAIL induced apoptosis. mTOSO stably transfected Jurkat cell clone TF-12, control Jurkat cell clone BC-7 which is stably transfected with vector only and parental Jurkat cells were treated with various amount of human recombinant TRAIL for 16 hrs. The survival rate was determined by FACS analysis of cells stained with Propidium Iodide (PI) and Annexin V conjugated with FITC, as described in Material and Method. The results shown were the average of three experiments. 62 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.5 Discussion Apoptosis is an essential process during the development of the immune system and for the maintenance of T- and B-cell homeostasis. In this study, we cloned and characterized the mouse homologue of a human anti-apoptotic TOSO gene (Hitoshi et al., 1998). The 422 amino acids mouse TOSO has 56% homology with the 390 amino acids human TOSO. The analysis of Kyte-Doolittle hydropathy plot displayed striking similarity of mouse TOSO to human TOSO, both having a hydrophilic leader sequence and a transmembrane domain (Fig. 2-2). The tissue expression pattern of mouse TOSO was similar to that of human TOSO in that the transcripts were both detected in heart, lung, thymus and spleen. In addition, CD3+ and CD19+ mouse B cells and CD4+ and CD8+ mouse T cells from B6 strain were found to express mTOSO, which was consistent with lymph node and peripheral blood leukocytes expressions of human TOSO (Hitoshi et al., 1998). Furthermore, two larger transcripts (3.8 kb and 5.8 kb) with lesser abundance were detected in mouse spleen tissue. Similarly, human TOSO transcripts in spleen also showed three larger transcripts (2.8, 3.5, and 4.3 kb) in spleen and lymph nodes (Hitoshi et al., 1998). These larger transcripts might be the result of alternative splicing in these tissues or the products of incomplete processed messages. It could also be the results of non-specific binding of mTOSO with other spleen specific genes. However, the expression was not detected in kidney or liver in mice as in human counterparts. The cDNAs of TOSO of several normal and autoimmune diseased mice strains were sequenced and sequence variations were found in these various strains. Whether they are mutations or polymorphrism remains to be determined (Fig 2-3). 63 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The computer analysis suggested that, as its human homologue, mTOSO is a type I membrane protein. In the study of human TOSO, the subcellular localization of TOSO was only deduced by water-soluble cross-linker experiments (Hitoshi et al, 1998). To directly demonstrate the cell membrane localization of mouse TOSO in an unambiguous approach, we conducted immunostaining assay where the c-terminal HA-tagged mTOSO was visualized by confocal microscopy (Fig.2-5). Later deletion mutation studies of mTOSO by in vitro protein binding assays confirmed that mTOSO is indeed a type I receptor where its C-terminal is involved in the binding (Fig. 2-10D). To further determine whether mTOSO is a type I membrane protein or not, FACS analysis can be used to sort out non-permealized cells recognized by anti-HA antibody. The inability of anti-HA antibody to recognize the cytosolic C-terminal HA-tagged cells will indicate that mTOSO is indeed a type I receptor, otherwise, it is a type II receptor. To study the function of mouse TOSO, we established stably transfected Jurkat cell lines. The expression of the transfected mouse TOSO gene was confirmed by RT-PCR using specific primers (Fig. 2-6A). Clones that contain transfected mouse TOSO gene significantly protected cells from FasL-induced apoptosis compared to the control Jurkat cells (Fig. 2-6B). Similarly, vector only transfected clones showed similar sensibility to FasL as Jurkat cells (Fig. 2-6B). This result not only revealed that we had successfully cloned functional mouse homologue of TOSO, but also, it unequivocally demonstrated that mouse TOSO inhibits apoptosis induced by FasL. Since the Fas pathway share some fators 64 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mediating the signal transduction in the pathway with TNFa singaling pathway, our results showed that mouse TOSO also protects cells from TNFa-induced apoptois (figure 2-7). Since caspase-8 is the apical molecular in this pathway, we therefore examined whether the activation of this molecule was somehow repressed upon apoptosis triggering. Not surprisingly, the caspase-8 activity in Jurkat cells over-expressing mouse TOSO was dramatically inhibited comparing to control Jurkat cells that do not express mouse TOSO, as indicated by enzyme activity assay (Fig. 2-8). In agreement with our results, human TOSO also prohibited pro-caspase- 8 from being processed into active form as demonstrated by Western Blot assay (Flitoshi et al., 1998). Based on RT-PCR analysis by Flitoshi et al., it was postulated that human TOSO inhibit caspase-8 activation through increasing mRNA level of cFLIP, which disturbs DISC formation (Scaffidi et al., 1999). However, rapid assembly of DISC complex which is detectable 60 minutes after Fas-induction in Jurkat cells and within 30 minutes in SKW6.4 B lymphoblastoid cells (Scaffidi et al., 1998; Scaffidi et al., 1999) argue against such hypothesis for the prolonged processes of transcription and translation of cFILP. In contrast, a faster response mechanism in which a pre-exist factor TOSO inhibits Fas-induced apoptosis through the disruption of DISC complex formation and consequently inhibits caspase-8 activation is more likely to be the modus operandi. In support of this hypothesis, immunostaining assay of mTOSO implies the co-localization of mTOSO with the control cell surface marker Fas (figure 2-5). In addition, in vitro protein binding assay demonstrated the specific binding of mTOSO to FADD. To explicitly demonstrate how mTOSO 65 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. disrupts DISC formation, co-immunoprecipitation of DISC complex experiments remain to be conducted. Nonetheless, the direct protein-protein binding at DISC hypothesis proposed by this study does not exclude the increased cFLIP transcription hypothesis as proposed by Hitoshi et al.(1998). To get a broader spectrum in our understanding of mouse TOSO in apoptosis, it is of interest to examine its function in protecting cell death triggered by other apoptotic stimuli, such as TRAIL and TNF, besides the study Fas-induced apoptosis. Our results indicated that ectopic expression of mouse TOSO in Jurkat cells does not protect cells from TRAIL induced apoptosis, comparing to control cells which do not express exogenous mTOSO (figure 2-11). Apparently, this result is against the expected protection function by mTOSO because of the involvement of FADD and caspases in the currently known TRAIL pathway (Masters et al., 1996; Mariani et al., 1997; Mariani and Krammer, 1988) and its apparent involvement in the DISC formation illustrated by this study. However, ectopic expression of FADD-DN in amounts sufficient to block Fas-induced cell death did not block apoptosis triggered by TRAIL. This indicates that a FADD-independent pathway links TRAIL to caspases (Masters et al., 1996). Furthermore, cells from FADD-deficient mice showed full responsiveness to DR4; on the contrary, induced by Fas, TNFR1 and DR3 showed resistance, confirming the existence of a FADD-independent pathway that couples TRAIL to caspases (Yeh et al., 1998; Zhang et al., 1998). In addition, one more layer of complexity is added by the fact that except Fas, other TNF family receptors depend on other intermediate proteins, rather than the direct binding to 66 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. FADD. For instance, TNFR1, DR3 and DR6 bind to intermediate protein TRADD, which functions as the docking site for FADD (Ashkenazi and Dixit, 1998; Wallach et al., 1999). In particular, DAP3 is the intermediate protein which links DR4 and DR5 to FADD in TRAIL-induced apoptosis (Miyazaki and Reed, 2001). Therefore, this result is in support of the notion that multiple pathways involving other yet to be determined factors exist in TRAIL triggered apoptosis. In summary, our study demonstrated that type I cell membrane mouse protein TOSO can protect T cells from fasL induced apoptosis by inhibiting caspase-8 activation. One of the potential mechanisms is to disrupt formation of functional DISC demonstrated by its binding to FADD molecular, a component of DISC complex. The anti-apoptotic function of mTOSO was further supported by experiments in primary T cells with over-expression of this gene. Primary T cells in mTOSO transgenic mice showed enhanced resistance upon FasL stimulation comparing to that of none-transgenic mice. Numerous studies have demonstrated the linkage of apoptosis with autoimmune diseases. The most striking example is that mouse lacking functional Fas (lpr)(Watanabe-Fukunaga et al., 1992) or FasL (gld) (Takahashi et al., 1994) develops severe autoimmune lymphoproliferative disorders. It was of interest to determine the effect of over-expression of mTOSO on the immune system in mice. As described in details in chapter III of the transgenic studies of mTOSO, founder transgenic mice with T-lymphocyte targeted expression of mTOSO under lek proximal promoter were generated on B6DBAF1 background. To determine whether overexpression of mTOSO can exacerbate the disease in 67 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. autoimmune mice, the founder mice were then back-crossed onto NZM2328 lupus mice background. The back-crossed mice were selected by microsatellite markers of B6 and NZM2328. In transgenic mice with more NZM2328 genetic background than B6 background, early on-set as early as one month old was observed, comparing to the development of the syndromes at well over six month of NZM2328 lupus mice. This strongly suggests that over-expression of mTOSO most likely exacerbate the autoimmune disease in NZM2328 lupus mice. To clearly illustrate this point, phenotype analyses of mTOSO transgeneic mice on pure NEM2328 is required. Nonetheless, primary T cells from spleens of back-crossing mTOSO transgenic mice, normal B6 mice and lupus NZM2328 mice were subjected to FasL induced apoptosis assays (Chapter III, Figure 3-4B). These results indicated that T cells from mTOSO transgenic mice with the higher expression of the mTOSO gene (Chapter III, figure 3-4A) were more resistant to apoptosis than T lymphocytes from non-transgenic mice, B6 mice and NZM2328 mice. All these results strongly suggest that mTOSO is a critical factor in the regulation of aopotosis. Future experiments in transgenic mice studies will further illustrate its important regulatory role in apoptosis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER III TRANSGENIC STUDIES OF mTOSO WITH TARGETTED T-CELL EXPRESSION 3.1 Abstract Gain- and loss-of-function models of genes in the core apoptotic pathway indicate that the deregulation of cellular homeostasis can be the primary pathogenesis of diseases. Studies have suggested that insufficient apoptosis can result in autoimmunity or cancer, whereas accelerated cell death leads to degenerative diseases, immunodeficiency and infertility. Accumulating evidence suggests apoptosis is playing vital roles in autoimmune diseases. To study the effect of mTOSO on the balance of autoimmunity and immune tolerance when over expressed in transgenic mice, in this study, mTOSO was expressed specifically in T cells under murine lek proximal promoter. The mTOSO transgene was backcrossed onto NZM2328 autoimmune background and the transgenic mice showed earlier onset of the autoimmune symptoms. Treatment of the primary T lymphocytes from mTOSO transgenic mice with FasL showed higher resistance to apoptosis, comparing to primary T cells from non-transgenic littermates. Future analyses of the transgenic mice on the cellular level, pathological analyses will yield more insights of how mTOSO functions in autoimmune diseases. Furthermore, it would be of interest to examine whether down regulation of mTOSO could ameliorate the symptom or inhibit the onset of the diseases by injecting anti-mTOSO antibody therapy or administering mTOSO RNAi. This would not only confirm the critical 69 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. role mTOSO played in immune system, but also, it would provide a future clinic therapy model for human patients. 70 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.2 Introduction 3.2.1 Autoimmune Diseases Apoptosis plays an important role in autoimmune diseases (Mullauer et al., 2001). Studies in the past several years have provided significant evidence that the apoptotic cell plays a central role in tolerizing B cells and T cells to both tissue specific and ubiquitously expressed self-antigens, and may drive the autoimmune response in systemic autoimmune disease. Fas-mediated apoptosis is an essential mechanism for maintenance of immune homeostasis and plays important role in autoimmune diseases. Abnormally increased or decreased Fas-mediated apoptosis is a major pathogenic mechanism of several diseases, including systemic or tissue-specific autoimmune disease and immune deficiency. Mice Qpr) carrying recessive mutations in Fas ,where a transposable element insertion inactivates FAS transcription, developed systemic autoimmunity and lymphoproliferation (Watanabe-Fukunaga et al., 1992; Chu et al., 1993). Similar syndrome is observed in gld mice with recessive FasL gene mutation (Takahashi et al., 1994). Fas transgenic mouse under CD2 promoter has demonstrated the importance of controlling Fas-mediated apoptosis by restoring apoptosis function and ameliorated autoimmune symptoms (Wu et al., 1993). It has been reported that mutations in Fas associate with human lymphoproliferative syndrome (Rieux-Laucat et al., 1995) and autoimmune lymphoproliferative syndrome (Fisher et al., 1995). Fas gene mutations have been detected in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with 71 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. autoimmunity (Drappa et al., 1996). The autoimmune lymphoproliferative syndrome (ALPS) is clinically characterized by massive lymphadenopathy and splenomegaly, which typically presents in childhood (Sneller et al., 1992; Sneller et al., 1997). The syndromes also include autoimmune hemolytic anemia, thrombocytopenia and other autoimmune manifestations (Watanabe-Fukunaga et al., 1992). ALPS patients also have expansion of an unusual circulating population of CD4CD8' T cells, which express a(3 TCR that are normally found in a very low level. In addition, patients have large number of circulating B cells together with polyclonal hypergammaglobulinemia. These findings are similar to those found in Fas deficient (Ipr) and Fasl (gld) mice (Cohen et al., 1997). In Hashimoto's thyroiditis, the infiltration of activated T cells and suicidal FasL-positive thyrocytes cause Fas-mediated apoptosis of thyrocytes. In systemic lupus erythematosus (SLE), the tolerance breakdown of self-antigens leads to the development of autoantibodies. In Rheumatoid arthritis (RA), which is characterized by pronounced hyperplasia of the synovial tissue, cell infiltration and periarticular osteoporosis, the reduced sensitivity to apoptosis in synovial cells and infiltration of autoreactive T and B cells contribute to the formation of the disease. These data suggest that apoptosis may be implicated in the pathogenesis of autoimmunity, whereas the mechanisms might be distinct in each autoimmune disease (Eguchi, 2001). Apoptosis also play an important role in Systemic Lupus Erythematosus (SLE). SLE is a multisystem autoimmune disease characterized by high level 72 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. autoantibodies against autoantigens. These autoantigens include nucleosomes, splicing ribonucleoproteins, phospholipid/phospholipid-binding protein complexes, and ribonucleoprotein particle Ro/SSA and La/SS-b. Patients with SLE have tissues and cells damaged by deposition of pathogenic antibodies. Generally, SLE patients have abnormal B- and T-cell functions. The clue of apoptosis playing a role in SLE came from the finding that diverse autoantigens all clustered and concentrated in and on the surface blebs of apoptotic cells (Casciola-Rosen et al., 1994), which is supported by numerous studies (Miranda-Carus et al., 2000; Price et al., 1996; Casciola-Rosen et al., 1996; McArthur et al., 2002). It is proposed that the apoptotic cell surface is an active B-cell tolerogen in vivo, a property implicating for explaining why defects in apoptotic signaling and execution are associated with systemic autoimmunity. Accumulating evidence supports the concept that defects in normal tolergenic clearance of apoptotic cells may be one factor that predisposes an individual to the development of systemic or tissue-specific autoimmunity (Voll et al., 1997; Ronchtti et al., 1999; Huynh et al., 2002; Cohen et al., 2002). Tremendous evidence has indicated intimate relation between tolerogens and self-immunogens, where, depending on additional contextual signals, striking outcome can be elicited by the same antigens. It has been proposed that apoptotic cells act as repositories of such antigenic source. Under normal homeostatic conditions, the apoptotic cell appears to be a potent tolerogen to induce editing of B cells recognizing apoptotic cell surface, accessing immature dendritic cells traveling to lymph nodes, tolerizing T cells and inducing the secretion of anti-inflammatory cytokinesis. Under diseased 73 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. conditions, autoantigens in and on apoptotic cells induce autoimmunity, potentially through abnormal apoptotic signaling and effector pathways, decreased clearance, or abnormal signaling thresholds on responding lymphocytes. 3.2.2 TOSO Transgenic Mice Transgentic and knockout models support the notion that apoptosis is playing a vital role in autoimmune diseases. Lpr (lymphoproliferation) and gld (generalized lymphproliferative disease) mice, which are loss-of-function mutant mice of Fas and Fas Ligand (FasL), respectively, show that these mutations are responsible for the early onset of systemic disease in MRL mice. These data suggest that autoreactive immunocytes are eliminated by the function of Fas/FasL. With the knowledge of the relationship between apoptosis and autoimmune diseases, numerous clinical trials targeted at reversing the imbalance between apoptosis and self-tolerance have been implemented. The success of these trials further supports that disruptions of normal apoptosis process will cause or contribute to human immune diseases. Since mTOSO is highly expressed in lymphoid cells, T- and B-cells are key players in maintaining immunity, it is of interest to study the effect of mTOSO on the balance of autoimmunity and immune tolerance when overexpressed in transgenic mice. In this study, mTOSO was expressed specifically in T cells under murine lck proximal promoter (Chaffin et al., 1990). Because autoimmune disease is caused by involvement of multiple genes, the analysis of mTOSO transgene on autoimmune background, such as NZM2328 would likely to yield discernible 74 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. effects. Our preliminary data has shown that mTOSO transgenic mice showed earlier onset of the autoimmune symptoms, comparing to NZM2328 mice. 75 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.3 Materials and Methods Generation lck-TOSO Vector: mTOSO cDNA was inserted into the BamI site of the pl017 expression vector containing the lck proximal promoter (Chaffin et al., 1990). The 6.9 kb Notl fragment containing lck proximal promoter, mTOSO, and human growth hormone fragment was then injected into B6DBAF1 blastocysts and implanted into pseudopregnant mice. Genotyping of lck-TOSO Transgenic Mice: Founder mice were initially screened by PCR analysis of tail DNA using primers specific for human growth hormone gene hGH (5’-TAG GAA GAA GCC TAT ATC CCA AAG G-3’ and 5’-ACA GTC TCT CAA AGT CAG TGG GG-3’). The transgene-positive lines were further confirmed by Southern blotting analysis probed by 3 2 P labeled hGH fragment generated by PCR using the primers listed above (Random Prime Labelling, Amersham Parmacia Biotech). Mice Strain Generation: The founder mice on B6DBAF1 background were then crossed onto NZM2328 strain lupus background. The distribution of C57/BL-6 and NZM 2328 genetic background in the backcrossing mice was determined PCR assay by 47 micro satellite markers (Table 1). The PCR cycles are: step 1, 94° for 10 mins; step 2, 94° for 15 sec; step 3, 60° for 30 sec; step4, 72° for 40 sec; step 5, goto step 2 for 10 cycles; step 6, 89° for 10 sec; step 7, 60° for 30 sec; step 8, 72° for 40 sec; step 9, goto step 6 for 25 cycles; step 10, 72° for 10 min; step 11, end. 76 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Isolation and Treatment of Primary T cells: Primary T cells from transgenic mice were extracted from spleen and concentrated by passing nylon wool column. Briefly, freshly removed spleen was placed in petridish containing 10 ml complete RPMI-1640 [10% v/v FBS, lOOU/ml penicillin G, 100 pg/ml streptomycin, 1% v/v nonessential amino acids, 2 mM L-glutamine, lOmM HEPES buffer, and 50 pM 2-ME] (Invitrogen). With the plunger of a 1-ml syringe until mesh the tissue. The spleen cells were then washed 2X in complete RPMI, and subject to centrifugation with 1:1 Ficoll/RPMI (Atalanta Biologicals) at 800xg for 25 mins. The middle layer containing lymphocytes were extracted and washed 2X in complete RPMI. The cells were then transferred to pre-warmed 200 pm mesh nylon column. Incubate the column 45 min in the upright position in a 37°C, 5% CO2 humidified incubator. T-cells were then eluted in the first 15 ml fraction with warm complete RPMI. Primary T cells were then treated with or without various amount of soluble recombinant human Fas Ligand (Alexis) for different time periods. Cells were then washed in 1 X PBS, stained by Propidium Iodide (PI) and Annexin V conjugated with FITC in binding buffer (PharMingen), and subjected to FACS analysis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Tab) e 1: Sequences of microsatellite primers ScreeningMarkers Sense Primer Anti-sense Primer l D 1M IT3 tttttgttttcttttcttttccc ccctcttctggtttccacat 2 D IM IT 15 tccacagaactgtccctcaa atacactcacaccaccccgt 3 D 1M IT36 gaggaatgtagagtccaacctgg tgaatagattaagagcttggaagc 4 D 1M IT37 acaggacttcttactcaaaccacc ttcttttggcctctttggg 5 D 2M IT26 tgttctttgctcatccacca aggctgatggtaacagtggg 6 D 2M IT30 catccaagcagtaacgtagacg aaatgttacaccctctgcgg 7 D 2M IT175 atgacaaacaagaaataggaaggc catgtgcttgagcatgcac 8 D 3M IT129 caggggcttgatacagcagt acaatggcctggagatacatg 9 D 4M IT1 atgatgtacacttaggcattgca agaaatatggcaagcaaaatgg 10 D 4M IT 9 ggctttggaatgctatgcat tggcaggaggtatgacagaa 11 D 4M IT205 tgtgtgaacatgtctacccc ggggaccgaagtaacagtga 12 D 5M IT13 catcgttgctcttgacagga ccgggagaacccaaataagt 13 D 5M IT10 cgagaagttggaaagaccca ggcacccatgcctctatg 14 D 6M IT10 tcagaggaacaaagcagcat cctgtggctaacaggtaaaa 15 D 6M IT50 cagggagttccagactagcg ttggtctgatttgccttatgc 16 D7Nds4 gtgacaatacattcctgctgt ctcagatcttatctctagcac 17 D 7M IT12 gctgggtttattcattgcaa tccagctcatgggtagaaga 18 D 7M IT55 aaccccaatgagtcaatcatg caagacatagcagacgactgtacc 19 D 8M IT49 tctgtgcatggctgtgtatg tggtgtgctgctgatgct 20 D 8M IT94 gttggggctctgctctctc cacatatgcatacatatacatacacgt 21 D 8M IT104 gaaagcaaacttctaaaggattatatg tgcaaattctcaaactgataccc 22 D 9M IT12 attcaaggggcagtacacat tggtcctggtaaaactgcct 23 D 9M IT48 acttgacatttgcccaggtc cagatcagcttgtgcgctag 24 D 9M IT249 aagccctcttagaagtagtgtgtatg agccatgaactaacttacatgtatca 25 D 10M IT28 cctcctgtatgtgtatttaaagca ctgcccatctgaccctgata 26 D 10M IT35 gtgctgcttcacgtttgga caagcaaggtaaattggaaagg 27 D 10M T44 ccaccttagacagtttacatggc ccacgcccagcttacttct 28 D llN d s l taagaaccttctgtagttatt accttagttagagttggtctc 29 D 11M IT41 ctgctaaagtggggttaaatgc cgactgagcaagttgtatttctg 30 D 11M IT51 ccaaacagggtctgttttattc taacagggtgagtttagtgaaaca 31 D 13M IT78 acagcacgggtttatcatcc tatgcctgccaggcttctat 32 D 13M IT39 ggggacaggcactcttagca cacaaggcagactggtcaga 33 D 1 4 M IT 1 1 aatattttcatgtttggagtcgtg cactgcagtgtcaatttctacttt 34 D 14M IT18 aaggtggaccaggaaggagt gacaatgagagaccaaaaaatgc 35 D15M1T13 ggagacaaaaatgaactcctgg ttgtaagacaagcatagctcaaca 36 D 15M IT29 tcactctccacctcccagat gtctgctttgtcatatgctgg 37 D 15M IT41 gacacatggtcggttttatgg ctcctcctggaaaatgtcca 38 D 16M IT4 agttccaggctacttggggt gagccctcattgcaaatcat 39 D 16M IT79 atcctgcatgcttttgctct cagaggagagttgtctgtgttcc 40 D 17M IT10 tgcacttgcataaggaaaac gactttggggcctacttatg 41 D 17M IT42 gatcatctctgaatccccca ggacagaactggctccaaag 42 D 19M IT1 aatccttgttcactctatcaaggc catgaagagtccagtagaaacctc 43 M usC K M M ggaggttgcagtgaattcaag ccagaccatctgatccagatc 44 MusTSHB tgaataaaggactcctgagct tctgaagagtttgtcctcatc 45 T N F -A LP H A ggacagagaagaaatgggtttc ggcaatctggggccaatcaggaggg 46 TNF-R1 gagccaccagagaccaagaaa gccttagaggtagcaacaaaa 47 TNF-R2 aatcaggtaggacaggaagg gaacacttcatgtagccagg R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.4 Results Cloning Mouse TOSO gene was cloned into p i017 vector as decribed in the material and method. The transgene construct as shown in figure 3-1 was injected into B6DBAF1 mouse blastocysts . p1017 vector Notl BamHI Notl lck proximal promoter hGH mTOSO cDNA Figure 3-1. The construct of the lck-mTOSO transgene. Murine TOSO cDNA was inserted into the BamHI site of the pi 017 vector. This vector contains the mouse lck proximal promoter and human growth factor (hGH) gene. The 6.9 kb Notl fragment was injected to B6DBAF1 mouse blastocysts. Southern Blot Screening of Transgene Founder transgenic mice were screened by Southern Blot analysis to confirm the presence of mTOSO transgene in the chromosome as shown in Figure 3-2. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Founder Mice Lck-TOSO Vector Figure 3-2. Southern blot analysis of mTOSO transgenic mice. Tail DNA from transgenic pups were extracted and digested with EcoRI. 20 pg of digested DNA per sample was loaded in each lane. Lck-TOSO-hGH fragment used for blastocysts injection was used as positive control and DNA from B6 strain mouse as the negative control. The blot was probed by hGH fragment generated by PCR. PCR Screening of mTOSO Transgene The screening of mTOSO transgene was conducted by PCR analysis during the process of backcrossing of founder mice with NZM2328 mice as decribed in Material and Method. Screening of FI mice for TOSO transgene from NZW background crossed with mTOSO founder mice #8 and #20 on B6BDAF1 background is shown in figure 3-3. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Founder #8 X NZM2328 F1 Founder #20 X NZM2328 F1 1 2 3 4 5 6 8 9 10 11 12 B6 #8 1 2 3 4 5 6 7 8 9 B6 #20 2.0 Kb__ 1.0 Kb__ 0.5 Kb__ Figure 3-3. Screening of FI mice from NZM2328 background crossed with mTOSO founder mice #8 and #20 of B6BDAF1 background. Tail DNA from FI mice were extracted and hGH fragments which is fused with the transgene (-400 bp) were amplified by PCR using its specific primers. DNA from B6 mice was used as negative control; DNA from #8 and #20 founder mice were used as positive controls. mTOSO Protects Primary T Cells Isolated from TOSO Transgentic Mice from Apoptotsis To further confirm mTOSO’s anti-apoptotic function, the sensitivity to apoptosis in primary spleen T cells from transgenic mice was determined. The T cell-targeted mTOSO expression transgenic mice were established by injecting construct with mTOSO gene under /c#-promoter. The founder transgenic mice originally established on B6DBAF1 genetic background were then back-crossed to NZM2328 lupus genetic background. Primary T cells isolated from transgenic and non-transgenic mice on NZM2328 lupus genetic background were testeded for the sensitivities to FasL induced apoptosis. The expression of the mRNA level of mTOSO in transgenic mice, non-transgenic mice, B6 and NZM2328 mice were 81 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. determined by realtime PCR. The mRNA expression levels in transgenic and non-transgenic primary T cells were quantified by real-time PCR with (3-actin as reference gene. The data indicated that the expression levels of mTOSO in founder #8 transgenic mice were on average about 26-fold higher than that of the NZM2328 mice (Figure 3-4A). However, the expression level from founder #20 was about 2-fold as that of NZM2328 mice (Figure 3-4A). Treatment of the primary T lymphocytes from these mice with FasL revealed that cells from the founder #8 mTOSO transgenic mice had higher resistance to apoptosis, comparing to primary T cells from non-transgenic litermates (Figure 3-4B). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Realtime PCR Analysis of mTOSO Expression in Transgenic Mice 30 25 - < D _ ] C o *0) < / > 9 ? Q_ X LU o C O o I- E (D > _ r o 0 a: 20 15 - 10 □ B6 m NZM2328 □ 20Tg- □ 20Tg+ ■ 8Tg- □ 8Tg+ Mice Strains Figure 3-4A. Realtime PCR of mouse TOSO mRNA expression in B6 mice (n=6), NZM2328 (n=10), mice from #8 TOSO founder transgenic lineage (8Tg+, n=5), non-transgenic mice (8Tg n=5), mice from #20 TOSO founder transgenic lineage (20Tg+, n=3) and non-transgenic mice (20Tg-, n=6). The relative expression levels were normalized to that of NZM2328 mice. 83 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Spleen T Cell Death With FasL Treatment 100% n -------------------------------------------------------------------------------------------------------------------------------- T0S08(-) TOSO20(-) T0S08(+) TOSO20(+) Figure 3-4B. Primary lymphocytes from mTOSO transgenic mice (n=5) from #8 founder mouse line showed resistance to FasL induce apoptosis compare to none transgenic litermate (n-5). Student t-test showed the significance of this difference ( p = 0 . 0 0 0 2 ) . 84 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.5 Discussion Despite the fact that Fas and its signaling proteins are widely expressed, Fas functions quite differently in each cell type of the immune system. Fas triggers apoptosis in developing T cells in thymus. On the contrary, in vivo clonal deletion of most autoreactive T cells in the thymus does not require Fas. Fas pathway eliminates excess effector cells through a negative feedback mechanism called propriocidal regulation in mature T cells (Lenardo et al., 1995; Lenardo et al., 1999). T cell derived FasL can also induce apoptosis in B cells, APCs and target tissues to counter-balance the proliferation of active effector cells that could be damaging to the host in the course of an immune response against a pathogen. Fas signaling is highly regulated during mature T cell activation and differentiation, where naive T cells express little surface Fas, and TCR activated cycling T cells have increased Fas expression (Ju et al., 1995; Dhein et al., 1995; Zheng et al., 1995; Peter et al., 1997). The increased sensibility has been attributed to decreased level of apoptosis inhibitor cFLIP (Rafaeli et al., 1998). With unclear mechanism, the resistance of newly activated T cells to Fas induced apoptosis may promote unfettered T cell proliferation upon acute antigen exposure. Fas-induced apoptosis also enforces B cell self-tolerance, which may play a particularly important role in preventing autoantibodies secretion in Fas-deficient states (Lenardo et al, 1999). Unlike TCR-induced apoptosis in T cells, mature B cells do not appear to be sensitive to Fas-mediated apoptosis. Instead, it appears that the developmental state and stimulation of B cells determine whether or not B cells 85 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. are subject to Fas-induced apoptosis. Like mature T cells, elimination of autoimmune B cells does not require Fas (Goodnow et al., 1995). Treatment of the primary T lymphocytes from these mice with FasL indicated that cells from the founder #8 mTOSO transgenic mice had higher resistance to apoptosis, comparing to primary T cells from non-transgenic litermates. Further analysis of the transgenic mice on the cellular level and pathological analyses will yield more insights of how mTOSO functions in autoimmune diseases. mTOSO transgenic mice generated on NZM2328 background need to be characterized such as the tissue-specific expression of mTOSO protein, the histochemistry studies with various organs, embryonic and adult expression profiles of mTOSO gene to detect its correlation with the onset and severity of the diseases. The CD4 and CD8 subpopulation distributions at different stages of transgenic mice development need also to be analyzed. The comparison of sensibilities of primary T cells to activation induced cell death in transgenic and wild mice will give insights about the mechanism of mTOSO’s involvement in the pathogenesis of autoimmune diseases. In addition, it would be of interest to examine whether down regulation of mTOSO could ameliorate the symptom or inhibit the onset of the diseases by injecting anti-mTOSO antibody or administering mTOSO RNAi. This will not only confirm the critical role mTOSO in immune system, but also, it will provide a future clinic therapy model for human patients. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER IV SPLIT-UBIQUITIN SCREENING OF mTOSO BINDING PROTEINS 4.1 Abstract Interactions between proteins are among the most important events taking place in the cells, and the analysis of these interactions in vivo is essential for understanding their functions in the cellular context. Therefore, we undertook the in vivo screening process of potential interacting proteins using split ubiquitin system in collaboration with Dualsyetem Biotech Incorporation. In our screening system, mTOSO was fused with Cub-LexA-VP16 as the bait and adult mouse spleen library was linked to NubG as preys. After screening against histidine prototrophy and ( 3 galactosidase activity, a total of six positive clones were identified. Analyses of sequences of the six positive clones revealed three protein factors: 1-acylglycerol-3-posphate O-acyltransferase 3, eukaryotic translation elongation factor 1 beta 2, and Ca+ + -sequestering ATPase. The important role of calcium homeosatsis and invovlement of protein translation in the apoptotic process suggest potential functional interactions of these factors with mTOSO protein in its anti- apoptotic function. Further in vitro and in vivo protein binding studies will be needed to prove the binding of mTOSO with these factors and in vivo functional studies will yield insights of how mTOSO functions on the molecular level. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 4.2 Introduction Interactions between proteins belong to the most important events taking place in the cells, and the analysis of these interactions in vivo is essential for understanding their functions in the cellular context. With the recent sequencing of entire human genome, the need for thorough function analysis of the predicted 35,000 open reading frames has been highlighted. The identification of interacting proteins will be used to establish the so-called genome-wide interaction map in human genome, where to date, these maps have been established for the yeast Saccharomyces cerevisiae, the worm Caenorhabditis elegans and a pathogenic bacterium, Heliobacterpylori (Uetz et al., 2000; Walhout et al., 2000; Rain et ah, 2001). Traditional methods for detection of protein-protein interactions, such as co purification, or co-immunoprecipitation are not suitable for high-throughput screen in vivo. For this application, there are a few methods with various limitations, such as cross-linking of proteins with cel-penetrating reagents (Creighton, 1992) and use of resonance energy transfer between dye-coupled proteins micro-injected into cells (Adams et al, 1991). In addition, genetic approach has been taken to search for synthetic lethal or extragenic suppressor mutations which occur in genes whose products are associated with a protein of interest (Guarente, 1993). The most commonly used and powerful method is yeast two-hybrid system originally developed by Fields and Song in 1989 (Fields and Song, 1989). The yeast two-hybrid system takes advantage of the common property of transcription factors 88 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. where they possess both a DNA binding domain (DBD) and an activation domain (AD). In this system, the protein of interest X is fused to the DBD called “bait” and a cDNA library expresses proteins Y fused to AD called “prey”. The interaction of X protein and Y protein brings DBD and AD in proximity and results in the reconstitution of a functional transcription factor that consequently activates a reporter gene (Figure 4-1). Although yeast two-hybrid system is a proven method to identify protein-protein interaction with approximately 350 publications per year taking advantage of this approach, there is a major drawback with this system. Because the activation of reporter requires the localization of fused transcription factor to the nucleus, only the protein interactions that occur or can be reproduced in the nucleus are able to be detected. Therefore, this method excludes the detection of membrane proteins (Fields and Song, et al., 1989; Chien et al., 1991; Gyuris et al., 1993; Guarente, 1993). In addition, this method can not address the temporal aspects of the protein-protein interaction. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. MAS___________________ reporter gene I ' l IAS 1 ' I reporter gene X: protein of in terest, bait Y: potentially in teractin g protein o r cDNA library, prey I UAS I _______________ reporter gene Figure 4-1. Schematic representation of the yeast two-hybrid system. A: The two separated DBD and AD domains are not functional, therefore there is no expression of the reporter gene in a yeast reporter system. B: Fusion of two protein of interest X and Y with DBD and AD, respectively. No reporter gene expression is detected as X and Y proteins do not interact with each other. C: Interaction of X and Y proteins brings BDB and AD domain into proximity which restores its function. Consequently, the reporter gene is expressed. Recently, the Split-Ubiquitin System has been developed by Johnsson and Varshavsky (1994), which is capable of screening membrane protein interactions and has several advantages over other methods. Ubiquitin is a conserved protein with 76 residues and a single domain (3, S34). Amino acids 1-34 form the a-helix subdomain and the rest amino acids form the (3-sheet (Vijay-Kumar et al., 1987) 90 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. (Figure 4-2a). Ubiquitin plays a role in protein degradation by attaching to the N terminal of the proteins as a signal for their degradation (Varshavsky, 1997; Varshavshy, 1992; Rechsteiner, 1991). The ubiquitin-polypeptide junction is rapidly cleaved by ubiquitin-specific protease(s) (UBP), with the requirement of correctly folded ubiquitin moiety (Johnsson and Varshavsky, 1994; Baker et al, 1992). In the split ubiquitin system, a reporter which is a transcription factor, is attached at the C- terminal of ubiquitin and the cleavage can be visualized by the transcription of lacZ gene or his3 auxotroph gene. Insertion of a 68-peptide within the loop (amino acid 34-40) connecting the a-helix and p-sheet in ubiquitin does not interfere cleavage by UBPs (Figure 4-2b). Like the yeast two-hybrid system, the ubiquitin is divided and expressed in two parts, a N-terminal fragment (Nub/, amino acid 1-34, with I being isoleucine at position 13), and a C-terminal fragment (Cub, amino acids 35-76) fused with a reporter protein (Johnsson and Varshavsky, 1994). NubI and Cub-reporter assemble with strong affinity to form functional ubiquitin which can be cleaved by UBP (Figure 4-2c). Replacement of Ile-13 of wildtype NubI by glycine (NubG) or alanine (NubA) decreases the affinity between Nub and Cub, subsequently abolishes the cleavage by UBPs (Figure 4-2d). To reconstitute functional ubiquitin, additional protein contacts are needed, the interaction of protein PI and protein P2 that attach to NubG and Cub-reporter respectively can reconstitute ubiquitin and the detection of the cleaved reporter protein indicates interactions between protein PI and protein P2 (Figure 4-2e). 91 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. •UBP* m b p s H Figure 4-2. Schematic representation of the split-ubiquitin system, (a) Ribbon diagram of ubiquitin with its two subdomains. The arrow denotes the site of either a 68-amino acid insertion or a cut between the subdomains, (b) A newly formed ubiquitin moiety bearing an insertion (wary red line) between subdomains and linked to a reporter protein (Re). The correctly folded ubiquitin is cleaved by UBPs (lightening arrow), yielding the free reporter, (c) Separately expressed Nub and Cub can form quasi-native ubiquitin and the cleavage by UBPs is observed, (d) Single mutation at position 13 (denoted as Nm ub) abolishes cleavage by UBPs. (e) Interactions between protein PI and P2 which are linked to Cub-reporter and Nm ub, respectively, reconstitute functional ubiquitin recognizable by UBPs. Split ubiquitin system has been used to detect specific interactions between soluble and membrane proteins (John and Varshavsky, 1994; Dunnwald et al., 1999; Wittke et al., 1999; Stagljar et al., 1998). This system has several advantages: 1) in vivo and in situ detection of soluble and membrane protein-protein interaction, 92 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. because no nuclear localization is needed as in yeast two-hybrid system; 2) small modules Nub and Cub are attached to linker proteins, which minimizes potential steric hindrance; 3) kinetic and temporal analysis of in vivo protein interactions, which have previously been demonstrated using photocrosslinking in a cell-free system; 4) detection of transient interaction; 5) The detection of interactions mediated by cleavage by UBPs not by transcription in this system allows detection of proteins that themselves have transcriptional activity or repressing sequences, which are not suitable or undetectable in yeast two-hybrid system. The identification of interaction partners of membrane protein mTOSO would provide vital clues about its anti-apoptoic roles in the cells. Therefore, we undertook the in vivo screening process of potential interacting proteins using split ubiquitin system in collaboration with Dualsyetem Biotech Incorporation. In our screening system, mTOSO was fused with Cub-LexA-VP16 as the bait and adult mouse spleen library was linked to NubG as preys. After screening against histidine prototrophy and [3-galactosidase activity, a total of six positive clones were identified. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 4.3 Material and Methods Bait Cloning: mouse TOSO cDNA encoding full-length TOSO protein was cloned to vectors pCMBV4, pAMBV4 and pTMBV4 in frame with reporter gene LexA VP 16. These three vectors vary in the strength of the promoters, which allow express of mTOSO at different levels. The cloned constructs were transformed into E. Coli and plasmids were isolated, restriction enzyme digested and sequenced to verify that mTOSO gene was correctly inserted. Bait Expression: cloned mTOSO in bait vectors were transformed into yeast DSY-1 strain. Total cell extract and cell membrane fraction extract were made and Wester Blot analyses were performed by blotting with antibody against VP 16 domain of the reporter protein in the bail constructs. Control Experiments of Bait: In order to test for correct expression of mTOSO protein in yeast, mTOSO bait constructs were co-transformed with positive control plasmid pAlg5-NubI. Co-expression of Alg5-Nubl with mTOSO resulted in reconstitution of split ubiquitin. The activation of reporter genes, lacZ and his3, would happen only if the Cub-Lex-VP16 reporter moiety is present in the cytosolic side of the membrane. Yeast cells were selected by both X-gal assay and histidine prototrophy. Negative control plasmid pAlg5-NubG was also co-transformed with mTOSO bait. Yeast cells were selected on by both X-gal assay and histidine. 94 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Library Transformation and Screening for Interactors: The reporter yeast strain with mTOSO bait expressing was transformed with adult mouse spleen library. Clones were selected by both X-gal assay and histidine prototrophy. Plasmids were isolated from positive clones and retransformed in E. Coli. To identify true positive clones and eliminate false positive clones, the isolated plasmids were separately retransformed with mTOSO bait plasmid and control plasmid pMBV-Alg5. Only preys that yield a HIS+ LacZ? phenotype were selected and sequenced. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 4.4 Results Analyses of sequences of the six positive clones revealed three genes: l-acylglycerol-3-posphate O-acyltransferase 3 (Agpat3), eukaryotic translation elongation factor 1 beta 2 (Eeflb2), and Ca+ + -sequestering ATPase (Atp2cl). 4.4.1 l-acylglycerol-3-posphate O-acyltransferase 3 Three of the positive clones encode l-acylglycerol-3-posphate 0-acyltransferase 3 (Agpat3), which was first identified through generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences (Strausberg et al., 2002). It encodes an enzyme involved in lipid synthesis. Two other isomers have been studied. Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. 1-acyl-sn-glycerol-3-phosphate acetyltransferase , also called LPA acyltransferase (LPAAT), catalyzes the conversion of LPA to PA. By searching an EST database for human homologs of yeast LPAAT, West et al., (1997), identified cDNAs encoding 2 proteins which they designated LPAAT-alpha and LPAAT-beta. The predicted LPAAT-beta protein is 278 amino acids long. Overall, the sequences of the 2 human proteins are 12% identical to the sequence of yeast LPAAT. Northern blot analysis revealed that, unlike LPAAT-alpha, LPAAT-beta is expressed in a distinct tissue-specific pattern, with the highest levels of expression observed in liver and 96 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. heart. West et al., (1997) demonstrated that both human LPAATs complemented an E. coli LPAAT mutation. Overexpression of the human LPAATs in mammalian cell lines led to increased enzyme activity. This increase in activity correlated with enhanced transcription and synthesis of IL6 and TNF-alpha, suggesting that LPAAT overexpression may amplify the cellular response to cytokine stimulation. Independently, Stamps et al. (1997) and Aguado and Campbell (1998) isolated LPAAT-alpha cDNAs. Aguado and Campbell (1998) reported that the sequence of the LPAAT-alpha protein shares 48% and 31% identity with those of LPAAT-beta and yeast LPAAT, respectively. They proposed that, based on sequence analysis and immunofluorescence studies, LPAAT-alpha is an endoplasmic reticulum (ER) transmembrane protein with 4 transmembrane domains. The potential active center of the enzyme is located between the third and fourth domains, facing the cytosolic part of the ER. Analysis of expression in insect and Chinese hamster ovary (CHO) cells suggested that a 58-amino acid signal sequence is cleaved from LPAAT-alpha to form a mature protein that migrates at 26 kD by SDS-PAGE. The recombinant protein has affinity for fatty acids of acyl-chain lengths of 12 to 18 carbons, with a slight dependence on the degree of saturation. 4.4.2 Eukaryotic translation elongation factor 1 beta 2 Elongation factor-1 is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. EF1 is a major protein of eukaryotic cells and exists as a 97 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. heterotrimer consisting of the subunits EF1-alpha (130590), EFl-beta, and EFl-gamma (130593). There is also an EFl-delta (130592), a form homologous to EFl-beta. Hence, 2 species of EF1 with composition EF1-alpha/beta/gamma or EF1-alpha/delta/gamma can be formed (van Damme et al., 1990). EF1 comprises 2 distinct functional domains: a nucleotide binding domain provided by EF1-alpha and a nucleotide exchange protein complex EFl-beta/gamma. 4.4.3 Ca(2+)-transporting ATPase 2C1 Hailey-Hailey disease (HHD; is an autosomal dominant disorder characterized by persistent blisters and erosions of the skin. By family linkage studies, the HHD region was localized to 3q21-q24. Study of a family carrying a deletion helped narrow the location. Hu et al. (2000) constructed a YAC/BAC contig from the centromeric breakpoint of the deletion to D3S1587, an interval of approximately 1.3 Mb. Within this region, it was found to reside an EST sequence that had been annotated as homologous to a yeast gene encoding a calcium ATPase. Because this gene was predicted to have a function related to that of SERCA2 (ATP2A2), Hu et al. (2000) isolated a full-length cDNA. Similar to other Ca(2+) ATPase genes, this gene encodes 2 alternatively spliced transcripts, ATP2Cla and ATP2Clb. These transcripts differed in their C termini (encoding amino acids 877 to the end), but had the same expression patterns in all tissues examined. ATP2Cla was predicted to encode 919 amino acids, and ATP2Clb was predicted to encode 888 amino acids. The protein encoded by ATP2C1 was highly homologous (97% 98 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. identity) to rat Pmrl, which in turn is homologous to the yeast calcium pump Pmrl, but less homologous to other calcium pumps. ATP2C1 is highly expressed in human epidermal keratinocytes and at various levels in other human tissues. Patients with HHD are not known to have extracutaneous manifestations of the disease. Hu et al. (2000) found no differences in ATP2C1 mRNA levels between skin taken from the axilla and skin from the buttock (sites particularly prone vs resistant to blistering, respectively, in HHD patients) of one normal individual and little change in ATP2C1 mRNA levels in normal human epidermal keratinocytes cultured with glucocorticoid. To screen HHD patients for ATP2C1 mutations, Hu et al. (2000) identified intron sites by comparison of genomic and cDNA sequences, designed primers flanking the 27 identified exons, and assessed PCR products from patients and controls by single-strand conformation polymorphism (SSCP) or conformation-sensitive gel electrophoresis (CSGE) analyses. Among 51 unrelated kindreds of European descent and 10 of Japanese descent, they identified 21 abnormalities (16/51 and 5/10). Of the abnormal sequences, 6 predicted single amino acid substitutions, 2 predicted aberrant splicing, and 13 predicted prematurely truncated products caused by frameshifts or single-basepair substitution. A high frequency of the last type of mutation supported a haploinsufficiency pathogenesis consistent with the complete deletion of the gene in 1 kindred and further suggested that calcium pumps of the PMR1 family function as monomers. The mechanism by which mutant ATPC1 causes acantholysis is unknown, but it may be through 99 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. abnormally elevated cytoplasmic calcium or abnormally low Golgi Ca(2+) levels. Elevated cytoplasmic calcium might act by altering posttranslational modification of proteins or by inducing changes in gene expression. Sudbrak et al. (2000) identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and nonconservative missense mutations, in ATP2C1 in patients with Hailey-Hailey disease. The identification of ATP2A2 as the gene defective in Darier disease provided further evidence of the critical role of Ca(2+) signaling in maintaining epidermal integrity. Ikeda et al. (2001) reported ATP2C1 mutations in 11 Japanese patients with Hailey-Hailey disease. Some affected individuals had unique clinical features (generalization of Hailey-Hailey disease and generalized skin eruption resembling keratotic papules in Darier disease), but other affected individuals did not, suggesting the presence of intrafamilial phenotypic variations. These findings reinforced the conclusion that differences in clinical phenotypes in Hailey-Hailey disease are probably related to factors other than the type of causative mutation. Chao et al. (2002) identified 7 different ATP2C1 mutations, 6 of them novel, in 7 Taiwanese kindreds with Hailey-Hailey disease. They found 3 deletion mutations, 2 nonsense mutations, 1 missense mutation, and 1 splicing mutation. Dobson-Stone et al. (2002) screened all 28 translated exons of ATP2C1 in 24 Hailey-Hailey disease families and 3 sporadic cases and identified 22 mutations (18 novel) in 25 probands. The novel mutations comprised 3 nonsense, 6 100 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. insertion/deletion, 3 splice-site, and 6 missense mutations, and were distributed throughout the ATP2C1 gene. Six mutations were found in multiple families investigated here or in the studies of Sudbrak et al. (2000) or Hu et al. (2000). Haplotype analysis revealed that 2 of these were recurrent mutations. Comparison between genotype and phenotype in 23 families failed to yield any clear correlation between the nature of the mutation and clinical features of Hailey-Hailey disease. The extensive interfamilial and intrafamilial phenotypic variability observed suggested that modifying genes and/or environmental factors may greatly influence the clinical features of this disease. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 4.5 Discussion There are many critical steps in controlling both intrinsic and extrinsic apoptotic pathways. Accumulating evidence has suggested that mitochondria and endoplasmic reticulum (ER) serve as important apoptotic control points where antiapoptotic Bcl-2 and proapoptotic BAX, BAK are located. The movement of Ca2 + from ER to mitochondria is a key process in activation of apoptosis by some stimuli (Ma et al., 1999; Pinton et al., 2001). Studies have shown that the amount of releasable Ca , rather than its concentration in the ER is the crucial requirement for transduction of the death signal (Nakamura et al., 2000). The antiapoptotic function 9 4" of Bcl-2 which is mediated through affecting Ca homeostasis provides a clue that • • 9 + mTOSO could also prevent apoptosis through interacting with the Ca ATPase ATP2C1. ATP2C1 is functionally related to SERCA, a calcium pump which is involved in Ca2 + homeostasis during apoptosis process (Pinton et al., 2000). Eukaryotic elongation factors have been shown to be concerned or likely to be concerned in various important cellular processes, including apoptosis. In Trypanosoma Cruzi, nuclear localization of E F-la has been observed in parasites undergoing apoptosis (Billaut-Mulot et al., 1996). In mouse 3T3 fibroblasts, increased expression of E F-la confers increased susceptibility to serum deprivation induced apoptosis. Furthermore, the suppression of EF-la by antisense E F -la RNA prevents the induction of apoptosis by serum deprivation. 102 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Further in vitro and in vivo protein binding studies will be needed to prove the binding of mTOSO with these factors and in vivo functional studies will yield insights of how mTOSO functions on the molecular level. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER V CONCLUSIONS AND FUTURE DIRECTIONS 5.1 Future Directions As mouse TOSO and its human homologue are novel genes which have not been studied in depth in various aspects, the following directions are suggested for future studies to yield better understanding of this important gene. 5.1.1 Molecular Mechanism Studies of mTOSO It would be of interest to study the in vitro and in vivo protein-protein interactions of mTOSO with other factor besides FADD protein, such as Caspase-8, Caspase-9, Fas, DR4, DR5, and TNFR1. The results from these studies will provide a more complete picture of how mTOSO inhibits apoptosis induced by Fas, TRAIL, and TNF. To confirm the binding of mTOSO with FADD and show physiological relevance of binding FADD and mTOSO, in vivo assays and mutagenesis assays would yield useful insights. 5.1.2 Transgenic Mice Studies of mTOSO mTOSO transgenic mice generated on NZM2328 background need to characterized such as the tissue-specific expression of mTOSO protein, the histochemistry studies with various organs, embryonic and adult expression profiles of mTOSO gene to detect its correlation with the onset and severity of the diseases. 104 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The CD4 and CD8 subpopulation distributions at different stages of transgenic mice development need also to be analyzed. The comparison of sensitivities of primary T cells to activation induced cell death in transgenic and wild-type mice will give insights into the mechanism of mTOSO’s involvement in the pathogenesis of autoimmune diseases. In addition, it would be of interest to examine whether downregulation of mTOSO could ameliorate the symptom or inhibit the onset of the diseases by injecting anti-mTOSO antibody or administering mTOSO RNAi. This will not only confirm the critical role mTOSO played in immune system, but also, it will provide a future clinical therapy model for human patients. 5.1.3 Promoter and Transcriptional Regulation Studies of mTOSO Gene To complete the understanding of mTOSO, it is necessary to study the regulation of mTOSO gene expression. Therefore, sequencing the promoter region of mTOSO gene is the fist step to conduct transcription studies. The analysis of potential transcription factors binding sites in the promoter region would provide insights of how mTOSO executes its function in autoimmune system. This would also provide possible targeting points for regulating mTOSO in pharmaceutical approaches. 5.1.4 Studies on the Cell Cycle-Dependent Regulation of mTOSO level in Fas-induced Apoptosis Tissue homeostasis is dependent on the proper relationship between cell 105 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. proliferation and cell death. As cells proliferate, cell cycle progression is tightly regulated by a network of positive and negative signals, and apoptosis is a highly regulated process to eliminate unwanted or defective cells. It is essential to properly regulate these coupled events since an imbalance between these two processes can lead to unwanted tissue atrophy or tissue growth. Growing evidence suggests that apoptosis is frequently associated with cells in the G1 phase of the cell cycle (king and Cidlowski, 1995; Meikrantz and Schlegel, 1995). Overexpression of a proapoptotic gene Bax in thymocytes is associated with a lowered level of p27 protein and accelerated p27 degradation as compared to the control cells. In addition, these cells enter S phase more rapidly, which correlates with early appearance of Cdk kinase activity. On the contrary, overexpression of the anti-apoptotic gene Bcl-2 results in delayed p27 degradation and delayed S phase entry by caspase 3 or caspase 3-like caspases, whose activation is, in part, determined by the balance between Bcl-2 and Bax (levkau et al., 1998). Most importantly, it has been demonstrated that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP. The finding of the possible mTOSO expression linkage with cell cycle certainly would provide explanations for increased TCR-induced apoptosis in the S phase of the cell cycle. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 5.2 Conclusion In summary, more studies in different directions need to be conducted in order to better understand this novel protein which plays an important role in autoimmune diseases. The results yielded from the above mentioned proposals would provide more insights into mTOSO’s functional mechanism. More importantly, the knowledge obtained from these studies would build foundations for future clinical therapies for human autoimmune diseases. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. APPENDEX A Inhibition of RNA Polymerase I Transcription in Differentiated Myeloid Leukemia Cells by Inactivation of Selectivity Factor 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.l Background The task of transcribing genes is carried out by three RNA polymerases in eukaryotes: RNA polymerase I (pol I), RNA polymerase II (pol II) and RNA polymerase III (pol III) (Roeder, 1976). Pol I synthesizes precursor 45S rRNA, which is processed into 28S, 5.8S and 18S rRNAs. Pol II transcribes genes that encode proteins. Pol III transcribes 5S rRNA, tRNA, 7SL RNA, U6 snRNA and a few other small stable RNAs, which are involved in RNA processing. The combined transcriptions of pol I and pol III exceed 80% of total RNA synthesis in growing cells. Pol I transcription takes place in discrete sites called nucleoli within the nucleus. rRNA is synthesized by pol I in the fibrillar centers, processed and assembled into ribosomes in the surrounding granular regions (Shaw and Jordan, 1995). This has long been regarded as a singularity of the pol I system, however, studies have shown that both pol II and pol III transcriptions are carried out at discrete locations (Pombo et al., 1999). In addition, at least part of the tRNA processing pathway may also occur in the nucleolus (Bertrand et al., 1998). Multiple copies of rRNA genes are found in tandem repeats in the nucleolus organizing regions on the chromosomes. The rRNA coding sequences are separated by the intergenic spacer (IGS) between the transcribed units. IGS contains a series of terminators, enhancers, a spacer promoter (SP), a proximal terminator (PT), the upstream promoter element (UPE), and the promoter core. Although the rRNA coding sequences are highly conserved, the sequences mediating transcription 109 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. initiation exhibits stringent (Grummt et al., 1982), though not absolute (Pape et al., 1990), species specificity. Despite little sequence similarity between rDNA promoters form different species, the overall structural organization of rDNA promoters are comparable from yeast to humans (Reeder, 1984; Moss and Stefanovasky, 1994; Paule, 1998; Planta, 1998). In general, the rDNA promoter consists of a start site proximal “core” promoter element (CORE) and distal elements, including the upstream control element (UCE), enhancers, terminators, and spacer promoters. CPE is located within a 39-bp region upstream of the transcription start site (Grummt, 1982; Yamamoto et al., 1984; Nagamine et al., 1987). The only conserved rRNA promoter sequence element within the core promoter called ribosomal initiator (rlnr) is an AT-rich, TATA-like sequence surrounding +1 (Perna et al., 1992). CPE is necessary and sufficient for faithful basal transcription, whereas the upstream elements stimulate promoter activity without affecting transcriptional specificity (Clos et al., 1986; Nagamine et al., 1987; Haltiner et al., 1986; Windle et al., 1986; Mougey et al., 1996). Upstream sequences enhance transcription both by increasing the stability of initiation complexes and by making more genes transcriptionally active (Schnapp et al., 1990; Osheim et al., 1996). The spacing and helical relationship of non-essential DNA which separates the core promoter and the UPE is critical. Half-helical turn insertions or deletions (+5 bp or + 15 bp) in this space severely compromised the activity of the promoter (Pape et al., 1990; Xie and Rothblum, 1992). Interestingly, the spacing between core element and UPE also play a role in determining species-specificity, as a 5-bp insertion converted Xenopus 110 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. promoter into a mouse specific promoter (Pape et al., 1990). Through the extensive studies using mouse-human chimeric promoters, it was showed that the species specificity resides solely within the core element (Learned et al., 1986). Upstream of UPE, the proximal terminator (PT) serves many functions including protecting the promoter from wandering polymerases (Bateman and Paule, 1988; Henderson et al., 1989), remodeling of chromatin over the promoter (Langst et al., 1998), and possibly, architectural folding of the rRNA repeats in the nucleolus (Sander and Grummt, 1997; Mason et al., 1998). IGS also contains repeated enchancer sequences that evolved from the spacer promoters by repetitive duplication and truncation (Pikaard, 1994). Mammalian pol I is a complex enzyme composed of at least 11 subunits (Hannan et al., 1998; Song et al., 1994). Two large polypeptides of 194 and 116kDa make up the structure core of the enzyme complex (Seither and Grummt, 1996; Seither et al., 1997). These two subunits contain homology to the ( 3 and (3 ’ subunit of bacterial RNA polymerases. Genetic and mutational studies analysis revealed that the conserved regions are involved in basic functions of RNA polymerses, i.e., transcription start site selection, initiation, elongation and termination (Archambault and Friesen, 1993; Died et al., 1995; Thuillier et al., 1996; Nudler et al., 1996). Several subunits of human RNA polymerase I have been identified, which showed high homology to yeast and mouse counterparts (Dammann and Pfeifer, 1998). In mammals, two basal transcription factors of pol I have been identified: the selectivity complex (SL1 or TIF-IB) and the HMG1 box architectural upstream 111 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. binding factor (UBF). Human SL1 contains four subunits, TBP and three TBP associated factors, TAF110, TAF63 and TAF48) (Comai et al., 1992; Heix et al., 1997). TBP-TAFs complex mediates species specificity of the rRNA promoters (Bell et al., 1989; Eberhard et al., 1993). Human UBF contains four high mobility group boxes, one of which mediates DNA binding and has a hyperacidic tail that is necessary for transactivation (Jantzen et al.,1990; Tuan et al., 1999). The NH2-terminal region has been found to mediate UBF dimerization (Jantzen et al., 1992). UBF is able to induce the enhancesome through three of its HMG1 boxes (Stefanovsky et al., 2001). Two enhancesomes may be formed at the pol I promoter, which explains the cooperative recruitment of SL1 to UCE and CORE. It is believed that UBF binds the promoter first, followed by the recruitment of SL1. It has been proven that the cloned components of SL1 are not sufficient for in vitro transcription (Paule, 1998). However, in presence of UBF, a strong cooperative DNA binding complex is formed at the rRNA promoter that is critical for initiation of transcription (Learned et al., 1986a, Bell et al., 1988). The recruitment of SL1 to the promoter is mediated by the COOH-terminal activation domain of UBF and is modulated by UBF phosphorylation (Tuan et al., 1999). Mutations in UBF that abolish DNA binding, such as the removal of the high mobility group box 1, or that impair the interaction between UBF and SL1, such as dephosphorylation or removal of the COOH-terminal domain, result in a drastic reduction in pol I transcriptional activity (Jantzen et al. 1992, Tuan et al., 1999; Voit et al., 1995). In addition, a recent study also showed that the interaction between 112 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. SL1 and UBF is influenced by the phosphorylation status of at least one of the TAFiS in the SL1 complex (Heix et al., 1998). Thus, these findings provide strong evidence that the network of interactions among UBF, SL1, and the rDNA promoter plays a major role in the regulation of pol I transcription. There is a large amount of evidence indicating that the regulation of protein synthesis is an important aspect of cell growth control. The cell growth rate is shown to be proportional to ribosome content (Kief and Warner, 1981). Studies from animal cells have demonstrated that the rate of growth is directly proportional to the rate of protein accumulation (Baxter and Stanners, 1978). Clearly, the availability of rRNA and tRNA plays a critical role in cell growth regulation as it was demonstrated that the synthesis of tRNA, rRNA and ribosome oscillates during the cell cycle (Stanner et al, 1979; Clarke et al., 1996; Rosenwald, 1996). In the following study, mechanism of the inhibition of pol I transcription in differentiated myeloid leukemia cells was determined. Our results indicated that SL1 is an important target for the regulation of pol I transcription during cell differentiation. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.2 Abstract Transcription by RNA polymerase I (pol I) regulates the rate of ribosome biogenesis and the biosysthetic potential of the cell; therefore, it plays an important role in the control of cell growth. Differentiation of the human promyelocytic leukemic cell line U937 is accompanied by drastic decreases in pol I transcriptional activity. We have used cell-free extracts prepared from undifferentiated and differentiated U937 cells to investigate the molecular mechanisms responsible for this inhibitory process. Our analysis indicates that the activity of the TATA binding protein (TBP)/TBP-associated factor (TAF) complex selectivity factor 1 (SL1), one of the factors required for accurate and promoter-specific transcription by RNA pol I, is severely repressed in differentiated U937 cells. Moreover, the reduction in SL1 activity is nor a consequence of a decrease in SL1, because there is no detectable difference in the abundance of TBP or TAFs before and after U937 cell differentiation. In conclusion, our results indicated that the selectivity factor SL1 is an important target for the regulation of pol I transcription during cell differentiation. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.3 Introduction During hematopoietic cell differentiation, quantitative and qualitative changes in gene expression occur, and disruption of the balance between proliferative and antiproliferative signals can lead to the abnormal growth associated with leukemia and other neoplastic disorders. The underlying regulatory mechanisms that are involved in these processes are poorly understood. The human promyelocytic leukemic cell line U937 is an established model for studying hematopoietic cell differentiation in vitro (Harris et al., 1985). These immature cells can be induced by the phorbol ester TPA to differentiate along the monocytic lineage into functionally and morphologically mature nonproliferating cells. Because a large amount of the cell's energy and resources during cell growth and cell division are used to make ribosomes, regulation of rRNA synthesis and ribosomal biogenesis may provide an important mechanism for controlling these cellular processes. Thus, molecules that modulate the expression the rRNA genes may exert a dual effect on both cell growth and cell division (Polymenis and Schmidt, 1999). rRNA genes are transcribed by a specialized polymerase, RNA pol I, which is localized in the nucleoli of eukaryotic cells (Paule, 1998; Grummt, 1999; Reeder, 1999). At least two factors, UBF and SL1, in addition to RNA pol I, are necessary to direct accurate and promoter-specific initiation of transcription from the rRNA gene promoter (Learned et al., 1986a; Learned 1986b). Human UBF is a Mr 97,000 polypeptide that recognizes both the core and upstream control elements of the 115 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. human rRNA promoter in a sequence-specific manner (Bell et al., 1988; Jantzen et al., 1990). Human UBF contains four high mobility group boxes, one of which mediates DNA binding and has a hyperacidic tail that is necessary for transactivation (Jantzen et al., 1992; Tuan et al., 1999). The NH2 -terminal region has been found to mediate UBF dimerization (Jantzen et al., 1992). The second essential factor necessary for accurate RNA polymerase I transcription is the selectivity factor SL1. SL1 is a multisubunit complex composed of TBP and three TAFs, TAFi48, TAFi63, and TAFil 10 (Comai et al., 1992; Comai et al, 1994; Rudloff et al., 1994). SL1 does not bind specifically to the rRNA promoter. However, in presence of UBF, a strong cooperative DNA binding complex is formed at the rRNA promoter that is critical for initiation of transcription (Learned et al., 1986a, Bell et al., 1988). The recruitment of SL1 to the promoter is mediated by the COOH-terminal activation domain of UBF and is modulated by UBF phosphorylation (Tuan et al., 1999). Mutations in UBF that abolish DNA binding, such as the removal of the high mobility group box 1, or that impair the interaction between UBF and SL1, such as dephosphorylation or removal of the COOH-terminal domain, result in a drastic reduction in pol I transcriptional activity (Jantzen et al. 1992, Tuan et al., 1999; Voit et al., 1995). In addition, a recent study also showed that the interaction between SL1 and UBF is influenced by the phosphorylation status of at least one of the TAFis in the SL1 complex (Heix et al., 1998). Thus, these findings provide strong evidence that the network of interactions among UBF, SL1, and the rDNA promoter plays a major role in the regulation of pol I transcription. 116 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. RNA pol I activity is tightly linked to the signals that control cell growth (Paule, 1998; Grummt, 1999), and a number of physiological and pathological stimuli affect the rate of RNA pol I transcription (Grummt et al., 1976; Hammond et al., 1988; Cavanaugh and Thompson, 1986; Mishima et al., 1979; Zhai et al., 1997). Recently, it has been proposed that the retinoblastoma tumor suppressor gene product (pRb) may be involved in the regulation of RNA pol I transcription in human cells, which are induced to differentiated by the addition of TPA (Cavanaugh et al., 1995). These studies showed that as soon as human myeloid cells U937 begin to differentiate, there is an accumulation of pRb in the nucleoli and a sharp decrease in rRNA synthesis (Cavanaugh et al., 1995; Rogalsky et al., 1993). In vitro experiments with mouse extracts and recombinant pRb suggested that the binding of pRb to UBF inhibits the DNA binding activity of this transcription factor (Voit et al., 1997). Despite these studies, it has not been proven that the interaction between pRb and UBF is indeed responsible for the repression of pol I transcription in differentiated U937. To define the mechanism of pol I transcription inhibition in differentiated cells, we have analyzed the transcriptional properties of extracts from U937 cells that were induced to differentiate by treatment with TPA. In this study, we found that the activity of the SL1 factor was severely inhibited in TPA-treated U937 cells, whereas UBF activity was not affected. Interestingly, using Western blot analysis and immnoprecipitation assays, we were able to determine that there was no significant difference in the abundance of the SL1 factor between mock and TPAinduced cells. Taken together, these results suggest that inhibition of SL1 117 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. activity, most likely through a prosttranslational modification of one or more of its components, is at the basis of the pol I transcription repression in differentiated hematopoietic cells. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.4 Materials and Methods Cell Culture: U937 cells were grown and subcultured every 2 days in RPMI 1640 supplemented with 10% heat-inactivated FCS at 37°C, 5% CO2 in humidified atmosphere. After seeding the cells in fresh growth medium at an initial density of 2- 3 X 105 cells/ml, TPA was added for 19 hr as a stock solution in ethanol to achieve the final concentration of 100 nM. Control cells (mock-induced) received an equal volume of ethanol. Hela S3 cells were grown in suspension in MEM supplemented with 5% newborn calf serum. Preparation of Cell Extracts: For the preparation of cell extracts from transcription assays, Western blots, and immunoprecipitations, we harvested each time -4-6 X 108 cells [-12-15 (150-mm) plates]. Whole-cell extracts were prepared accordingly to the method developed by Manley et al. (Manley et al., 1980). Nuclear extracts were prepared as described in Zhai et al. (21). Whole-cell extracts were used as starting material for the fractionation studies. Partially purified SL1 was prepared by chromatography on Poros HE1 (heparin-agarose). Briefly, whole-cell extracts were loaded onto a Poros HE1 column in TM buffer [50 mM Tris (pH7.9), 12mM MgCl2, 1 mM EDTA, and 10% glycerol] containing 0.1 KC1. The column was washed extensively with TM/0.1, and it was then step-eluted with TM buffer containing 0.3 M KC1, 0.4 M KC1, and 0.8 M KC1. RNA pol I eluted at 0.3 M KC1, UBF eluted at 0.4 M KC1, and SL1 eluted at 0.7 KC1. Transcription assays and 119 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Western blot analyses were used to identify fractions containing RNA RNA pol I,UBF, and SL1. Peak fractions for each activity were pooled and dialyzed in TM containing 0.1 M KC1 and 0.1% NP-40. Hela RNA pol I, UBF, and SL1 were prepared as described previously (Tuan et al., 1999; Comai et al., 1992). Protein concentrations were determined by Bradford assay. Transcription Assay: RNA pol I transcription assays were carried out as described previously using either 30 or 100 ng or rDNA gene template in the presence of 100 pg/ml of a-amanitin. Quantitation analysis was performed using a Phosphorimager (Molecular Dynamics). In the extract mixing experiment, the reaction mixture was incubated at 30°C for 15 min before the addition of all four ribonucleotides. RNA Purification and Analysis: Total RNA was isolated using a single step procedure by gaunidinium thiocyanate-acid phenol-chloroform extraction. Briefly, cells were collected and lysed in denaturing buffer [4M guanidinium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium lauryl sarcosine, and 0.15 M 2-mercaptoethanol], Genomic DNA was sheared by pipetting up and down several times, and total RNA was prepared by the addition of 2 M sodium acetate (pH 4.0), 1 volume of water-saturated acidic phenol, and one-fifth of chloroform: isoamyl achcohol (24:1). Samples were vortexed and centrifuged at 10,000 X g, and the RNA-containing aqueous phase was carefully collected. RNA was further precipitated by the addition of 2.5 volumes of ethanol, washed with 70% ethanol, air- 120 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. dried, and dissolved in diethyl pyrocarbonate-treated water. After quantitation by spectrophotometry, equal amounts of RNA were used for SI nuclease analysis. SI nuclease analysis was carried out using an oligonucleotide complementary to the region between -20 and 40 of the human rDNA gene that was labeled with 3 2 P at the 5’ end. Preliminary experiments were performed to assure that the radio-labeled oligonucleotide used in each reaction was in vast ( >10 fold ) excess over the target RNA. RNA pol I Assay: Random RNA polymerization assays were performed as described by Roeder (Roeder, 1974). Each reaction mixture contained 5 pg of nicked herring sperm DNA, lOOpg/ml a-amanitin, and 10 pg of protein extract. Antibodies and Immunoprecipitation Analysis: Rabbit polyclonal antisera raised against recombinant TBP, TAFs and UBF were affmity-perified accordignly to published procedures. Immunoprecipitation reactions were carried out as described by Comai et al. (Comai et al., 1992). Immunoreactivity was shown by the alkaline- phosphatase detection method. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5 Results A.5.1 rRNA Transcription Is Drasticaly Reduced after Differentiation of U937 Cells. To determine the level of rRNA synthesis before and after differentiation, total RNA was extracted from undifferented U937 cells and from cells that were induced to differentiate by treatment with TPA for 19 hr. Control cells were treated with the dame volume of ethanol, the solvent used to solubilize TPA. The level of 5’-precursor rRNA transcript in undifferentiated and differentiated cells was determined by SI nuclease protection assays. As shown in Fig. A-l, there is a dramatic difference in the abundance of 5’ rRNA between TPA-treated (Lanes 1 and 2) and mock-treated (Lanes 3 and 4) U937 cells. Quantitation analysis indicated that the level of pre-rRNA is reduced ~6-8-fold after cell differentiation, p-actin mRNA expression showed no variation between mock- and TPA- treated cells. Because the 5’ end of the precursor rRNA faithfully reflects the rate of initiation of transcription (Kass et al., 1990). Therefore, these results provide further evidence that pol I transcription is repressed after U937 cell differentiation (Cavanaugh et al., 1995). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TPA mock I 1! 1 2 3 4 SI profo# i M i — r i Figure A-1. RNA polymerase I transcription decreases after U937 cells differentiation. SI analysis of total RNA (5 pg) is isolated form TPA-induced (lanes 1 and 2) and mock-induced (lanes 3 and 4) U937 cells. Two independent preparations of total RNA from mock- and TPA-induced cells were tested. The RNAs were hybridized with a 5’ end 3 2 P-lableled 60 bases oligonucleotide complementary to the nucleotide -20 and +40 of the human ribosomal DNA gene. Quantitation by Phosphorimager indicates that transcription is approximately eight folds lower in differentiated (TPA-treated) cells (not shown). 123 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5.2 Extracts of Differentiated U937 Cells Are Impaired for pol I Transcription. To dissect the molecular events responsible for the down-regulation of transcription by RNA pol I in differntiated U937 myeloid cells, we then established an in vitro transciption system. Extracts were prepared from U937 cells that were either induced to differentiate with RTPA ant a concentration of 100 nM or mock- induced with an ethanol solution without TPA. Equal amounts of extracts were tested in an in vitro transciption assay using a plasmid containing the human ribosomal DNA promoter as template. Transcription assays were performed under the standard conditions, and rRNA transcripts were detected using the S1 nuclease assay (Ausubel et al., 1991). As shown in Fig. A-2, the RNA pol I transcriptional activity is ~6-fold lower in the TPA-induced U937 cell extract (Lane 3 and 4), as compared with the extract prepared from mock-induced cells (Lanes 1 and 2). These results demonstrate that the repression of rRNA synthesis upon TPA-induced differentiation of hematopietic cells is faithfully reproduced in an in vitro transcription system. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TP 4 WCEi U937 XJ937 « # * 1 2 3 4 Relative transcription a ctiv ity 1 2.5 | O 1 " T T j o 1 Figure A-2. Extracts from TPA-treated cells are deficient in pol I transcriptional activity. A human rDNA template (prHu3) was transcribed in vitro using 4 pg (lanes 1 and 3) or 6 pg (lanes 2 and 4) of whole cells extracts (wee) from undifferentiated (lanes 1 and 2) or differentiated (TPA-treated; lanes 3 and 4) U937 cells. Whole cell extracts were prepared as described in Materials and Methods. Transcription data were quantitated using a phosphorimager and relative activities are as indicated. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5.3 Extracts from Differentiated U937 Cells Do Not Contain a Soluble Repressor of pol I Transcription. To determine whether the decreased transcriptional activity was attributable to a soluble repressor found in the extracts from differentiated cells, we performed a mixing experiment. A constant amount of extract from undifferentiated U937 cells was mixed with increasing amounts of extracts from TPA-induced cells, incubated on ice for 30 min, and then tested in transcription reactions. As shown in Fig. A-3, the titration of increasing amounts of extracts from differentiated U937 cells (Lane 3-5) did not affect the transcriptional activity of the extracts from undifferentiated cells. These results exclude the possibility that the extracts from TPA-induced cells contain a soluble inhibitor of transcription and suggest that the inhibition of rRNA synthesis is most likely attributable to a specific decrease in the activity of one or more components of the pol I transcriptional machinery. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mock-U397 TPA-U937 -------------------II--------1 + TPA/U937 1 2 3 4 5 6 7 Figure A-3. Differentiated U937 cell extract does not contain a soluble repressor. A human rDNA template was transcribed in vitro using 5 pg of undifferentiated (mock-U937; lane 1-5) or differentiated (TPA-U937; lanes 6 and 7) U937 whole cell extract. In lanes 3-5, in creasing amounts (5 pg, 10 pg and 15 pg, respectively) of extracts from differentiated cells were added to reaction mixtures containing extracts (5pg) from undifferentiated cells and the rDNA template, and incubated at 30°C for 15 minutes before the addition of nucleotides. In lane 1, the rDNA template was omitted. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5.4 SL1 Activity Is Specifically Diminished in Differentiated U937 Cell Extracts. To determine whether the defect in transcription was attributable to an inhibition of the RNA pol I itself, we measured the pol I enzymatic activity in differentiated and undifferentiated U937 cell extracts. For this purpose, we used an assay that measures the ability of RNA pol I to randomly initiate transcription on nicked DNA. Several matched pair samples were tested, and in none of them was there any significant difference in the level of nucleotide polymerization by pol I between undifferentiated and differentiated extracts (Fig. A-4). In addition to RNA pol I, two additional factors, UBF and SL1, are required for accurate transcription of human rRNA genes. Regulation of either of these two factors may account for the observed repression of pol I transcription after differentiation. To test this hypothesis, we determined whether the addition of exogenous SL1 or UBF was able to rescue pol I activity in differentiated U937. SL1 or UBF fractions purified from Hela cells were added to in vitro transcription reactions containing extracts from TPA-induced cells. The results of these experiments indicated that SL1, but not UBF, was able to fully rescue RNA activity (Fig. A-5, A and B). To exclude the possibility that UBF added to the transcription reactions was nonfunctional, the transcriptional activity of Hela-purified UBF, using in vitro reconstituted transcription assays with purified SL1 and RNA pol I, is shown in Fig. 5C. Thus, differentiated U937 cell extracts appear to be primarily deficient in SL1 activity. Interestingly, the addition of purified SL1 could also stimulate the 128 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. transcriptional activity of undifferentiated U937 cell extract, suggesting that SL1 is a limiting factor for pol I transcription in U937 cells. mock-U937 Figure A-4. Undifferentiated and differentiated U937 cells extracts display similar levels of pol I polymerization activity. Non-specific RNA polymerase I assays were carried out as described in Materials and Methods using Hela, undifferentiated U937 and differentiated U937 whole cell extracts. Incorporated [3H]-UTP was counted in scintillation fluid with a Beckman scintillation spectrometer. Bars represent standard deviations calculated from three independent experiments. 129 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A Hela U937 TPA-U937 I II II-------------------------------------------1 + S L 1 + SM + U B F i i ^ 3 1 2 3 4 5 6 7 8 9 10 11 Figure A-5A. Exogenous SL1 can stimulate pol I transcription I differentiated U937 cell extract. In vitro transcription reaction were carried out using 4 ptg of whole cell extracts from Hela (lanes 1 and 2), undifferentiated U937 (lanes 3 and 4) and differentiated U937 (TPA-treated; lanes 5-11). Reactions in lanes 6, 8, 10 and 11 were supplemented with partially purified SL1 (lanes 6 and 8; 30 ng and 60 ng, respectively) or pure UBF (lanes 10 and 11; 10 ng and 40 ng, respectively). SL1 and UBF were purified from Hela cells, and their activity was assessed by transcription and footprinting assays (Tuan et al., 1999; Comai et al., 1992). These results have been reproduced in several independent preparations. In addition, identical results have been obtained using nuclear extracts. The arrow indicates the position of the protected oligonucleotide product. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TPA-U937 NXT 0 1 2 m 1 2 3 4 5 6 7 8 9 R elative transcription activity 1.3 1.0 1.3 2.3 3.2 4.7 3.1 4.5 6.2 Figure A-5B. Pol I transcription in differentiated U937 nuclear extracts can be stimulated by the addition of purified SL1. Transcription assays were performed using nuclear extracts from differentiated U937 cells (lanes 1, 4 and 7: 6 pg; lanes 2, 5, and 8: 9 pg; lanes 3, 6 and 9: 12 pg) and no SL1 (lanes 1-3), 1 pi (10 ng/pl) of SL1 (lanes 4-6) or 2 pi of SL1 (lanes 7-9). Identical results were obtained using whole cell extracts. Transcripts were quantitated using a phosphorimager and relative activities are as indicated. 131 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UBF - POL I + ........................- SL X + ► 1 2 3 4 Figure A-5C. Transcriptional activity of Hela-purified UBF. Transcription assays were performed using pol I and SL1 purified from Hela cells in the presence (lanes 2 and 3) or absence (lance 1 and 4) of Hela purified UBF used in the experiments shown in panel A. To confirm the deficiency of SL1 activity in TPA-induced U937 cells, we partially purified SL1 from undifferentiated and differentiated U937 extracts using a well-established fractionation procedure. The purified SL1 fractions were then tested in in vitro reconstituted transcription reactions in the presence of Hela-purified pol I and UBF. Importantly, the partially purified SL1 fraction did not contain any 132 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. detectable pol I and UBF activities (see “Materials and Methods”). As shown in Fig. A-6A, the SL1 fraction from differentiated (TPA-induced) U937 cells (lane 5-7) is several folds less active than the respective SL1 fraction from the undifferentiated cells (lane 2-4). In addition, to determine whether the activity of UBF also changed upon differentiation, we tested the partially purified UBF fraction from undifferentiated and differentiated cells in an in vitro reconstituted assay with SL1 and RNA pol I purified from either Hela or U937 cells (Fig. A-6B). UBF activity from both undifferentiated and differentiated U937 extracts is quite low compared with the activity of Hela-purified UBF. Interestingly, the partially purified UBF fraction from differentiated U937 cells was more active than the corresponding fraction from undifferentiated cells, as determined in both assays condition. These experiments have been carried out in the presence of limiting amounts of SL1 (0.1 ng) to maximize UBF response. These results indicate that UBF is not down-regulated upon U937 cell differentiation and further support the concept that a reduction in the activity of the SL1 factor is the major cause for the inhibition of rRNA synthesis in differentiated U937 cells. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. SL1 MocMJ. 937 TPA *11937 H e la m TPA RNA pol 1+ IjgjP 1 2 3 4 5 6 7 8 9 R eU < v « tr n nption activity 4.5 1.2 1.4 1.7 0.3 0.1 0.2 N O N O Figure A-6A. SL1 activity is repressed in differentiated U937 cell extracts. SL1 (0.8 M KC1 fraction) was purified by heparin-agarose column chromatography from Hela (lane 1, 50 ng), undifferentiated U937 (mock-U937; lanes 2-4; 50 ng, 100 ng and 200 ng respectively), and used in transcription reactions with 100 ng of a Hela fraction containing pol I and UBF. In lanes 8 and 9, SL1 from undifferentiated U937 (lane 8) or differentiated U937 (lane 9) was used in transcription reactions in the absence of pol I and UBF. Transcripts were quantitated using a phosphorimager and relative activities are as indicated. 134 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. M o c k IBdLa-POtl H tU S L ll TPA - ^ 1 ^ 1 m r + ----- 1 2 3 4 5 6 7 Rotative transcription activity 1.0 1 .7 i.i* 2,5 4.5 0.8 3.7 0.4 KC: vm-WLt a io c k m " f " ip1 Ilv L u P O L l H W f 8 9 10 II 12 Figure A-6B. UBF activity in U937 cell extracts. Top panel: UBF (0.4 M KC1 fraction) from undifferentiated (lanes 2, 3 and 6) or differentiated (lanes 4, 5 and 7) U937 cells was used in reconstituted transcription assays with pol I and SL1 purified from Flela cells. In lane 1, the Hela-purified pol I and SL1 were used in the absence of UBF. The arrow indicates the position of the protected oligonucleotide product. Transcripts were quantitated using a phosphorimager and relative activities are as indicated. Bottom panel: RNA pol I (0.3 M KC1 fraction; 6 pg/reaction) and UBF fractions [0.4 M KC1 fraction; 80 ng (lanes 8 and 10) and 250 ng (lanes 9 and 11)] from either undifferentiated (lanes 8 and 9) or differentiated (lanes 10 and 11) U937 extracts were used in reconstituted transcription assays in the presence of Hela- purified SL1. In lane 10, transcription was carried out with a Hela-purified fraction containing pol I and UBF, and Hela SL1. 135 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5.5 The Abundance of SL1 Does Not Change after Cell Differentiation. Human SL1 is a multiprotein complex comprised of TBP and three associated factors, TAFi48, TAFi63, and TAFillO. Several studies have indicated that each of these factors is necessary for the assembly of a functional SL1. To determine whether the reduction in SL1 activity in differentiated cells was attributable to a decrease in the abundance of one or more of its components, we carried out a series of Western blot analyses. Whole cell extracts from undifferentiated and differentiated U937 cells were resolved on SDS-PAGE gels, and the abundance of each of the four components of SL1 before and after differentiation was determined using antibodies raised against each protein. As shown in Fig. A-7A, there was no detectable change in the level of TBP, TAFi63, and TAFillO proteins after cell differentiation. In addition, the abundance of the UBF factor also did not change between undifferentiated and differentiated cells. The results strongly suggest that differentiation of U937 cells and inhibition of pol I transcription is not accompanied by a specific decrease in the level of any of the SL1 subunits. In addition, we observed an additional slower migrating band in the anti-TBP Western blot with differentiated extracts. To determine whether this band was phosphorylated form of TBP, we performed the Western blot analysis using extracts that were pretreated with alkaline phosphatase. As shown in Fig. A-7B, the slower migrating TBP band disappears in phosphatase treatment, suggesting that the diminished SL1 activity in differentiated cells may indeed result from posttranslational modifications of a SL1 subunit, such as TBP. 136 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mock TPA SLl mock TPA SLl -inti-'l \ I. | i 10 a»tf-TAPj«3 mock TPA SLl anfl-TAF^ mock TPA w rti-O T F Figure A-7A. Western blot analysis of TBP, TAFs and UBF from undifferentiated and differentiated U937 cells. 40 pg of mock-treated and TPA treated U937 extracts were resolved on a 10% SDS-PAGE and analyzed by western blot with antibodies as indicated. An SLl fraction from Hela cells was used as a control (SLl). All antibodies used were affinity-purified except for anti-TAFi48. .mock TP A A l* + - + ■ 1 2 3 4 and-TBP Figure A-7B. Western blot analysis of TBP from alkaline phosphatase-treated U937 extracts. 15 pg of mock-treated and TPA-treated U937 extracts were pre-incubated with 0.5 pi (1000 U/pl) of calf alkaline phosphatase (lanes 1 and 3) or 0.5 pi of phosphatase buffer (lanes 2 and 4) at 30 °C for 15 minutes, resolved on a 10% SDS-PAGE, and then analyzed by western blot with TBP antibodies. Arrow shows slower migrating TBP band. 137 mock TPA SLl aatf-TBP R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.5.6 TAFs and TBP Are Associated in an SLl Complex in Differentiated U937 Cells. Although each of the components of SLl was present in similar quantities before and after differentiation, the previous experiment did not address whether SLl was still present as a stable multiprotein complex after cell differentiation. For this purpose, SLl was immunoprecipitated from cell extracts prepared from an equal number of mock induced and TPA-induced cells. Affinity-purified antibodies raised against either TAFi63 or TAFillO were used to immunoprecipitate SLl. The immuno-precipitation products were then resolved on a SDS-PAGE gel, and SLl was detected using affinity-purified anti-TBP antibodies. As shown in Fig. A-8, no significant differences in the amount of TBP can be seen in the immunoprecipitation reactions, suggesting that SLl remains associated as a stable complex after differentiation. These results provide further evidence that the decrease in SLl specific activity is likely attributable to a posttranslational modification of one or more of its subunits. Of course, we cannot rule out that the decrease in SLl activity may be cause by the absence of the other SLl subunits, TAFi48, within the complex. Because of the low titer and poor quality of the antibodies against TAFi48, we were not able to obtain interpretable data in anti-TAFi48 immunoprecipitation reactions. Thus, we cannot strictly rule out that TAFi48 may be excluded from the SLl complex after cell differentiation. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. IP-. anti-TAf [ 110 IP; jwtt-TAFjM IP; pre-lntmune seru m mock TPA' *SU SM mode TPA fTBP mock TPA iTBP IgG hx. TBP -#** I 3 i 5 b 7 8 9 10 Western; anti-TBP Figure A-8. Immunoprecipitation of SLl from undifferentiated and differentiated U937 extracts. Equal amounts (approximately 3 mg of proteins) of mock-treated and TPA-treated U937 extracts were precleared with protein A-agrose and then incubated with affinity-purified antibodies against TAFillO (lanes 1 and 2) or TAFi63 (lanes 5 and 6). Immunocomplexes were precipitated by incubation with protein A-sepharose, resolved by SDS-PAGE, and analyzed by western immunoblotting with anti-TBP annfinity-purified antibodies. A partially purified SLl fraction (lanes 3, 4 and 10) and recombinant TBP (lane 7) were used as controls. Rabbit preimmune serum serum was used for the immunoprecipitations in lanes 8 and 9. 139 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A.6 Discussion TPA treatment of U937 human myeloid leukemia cells initiates a cascade of cellular events that profoundly influence the expression of a variety of genes and ultimately lead to cell differentiation. Differentiation of U937 cells is associated with a significant decrease in rRNA transcription. The TPA-induced repression of rRNA transcription is of particular interest because it directly affects ribosome production protein synthesis and therefore, provides a direct mechanism to control cells growth (Polymenis and Schmidt, 1999; White, 1997). To elucidate the molecular mechanisms underlying this repression of pol I transcription, we have analyzed the components of the transcriptional machinery before and after differentiation. Our studies indicate that the decrease in transcription reflects a drastic reduction in the activity of the selectivity factor SLl. Complementation assays and fractionation experiments reproducibly show that SLl activity is between 8-14 folds lower in differentiated than undifferentiated U937 cells. This is a significant finding that implies that regulation of SLl activity has an important role in the modulation of pol I transcription. Interestingly, Western blot analysis of SLl from extracts prepared from undifferentiated and differentiated U937 cells showed to appreciable difference in the abundance o any of the SLl subunits. Immunoprecipitation assays also show that in either extract, SLl is found as a stable multiprotein complex. These results suggest that SLl activity is most likely modulated by a posttranslational event induced by the differentiation process. The mouse orthologue of SLl, TIF-IB, has been shown recently to be the target of 140 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. regulation during mouse F9 embryonal carcinoma stem cell differentiation and the reduction in TIF-IB activity appeared to be associated with a decrease in the abundance of two mouse TAFs, mTAFi48 and mTAFi95 (Alzuherri and White, 1999). On the other hand, recent studies have indicated that mitotic inactivation of human rRNA synthesis is mediated by phosphorylation of TAFillO, one of the subunits of SLl (Voit et al., 1997). It is therefore tempting to speculate that the mechanism of human SLl inactivation in differentiated U937 cells resembles the mitotic process. It is currently unclear whether phosphorylation or dephosphorylation of any of the SLl subunits is involved in down-regulation of SLl activity in differentiated U937 cells. Preliminary experiments indicate that a slower migrating form of TBP, present in the extracts from differentiated U937 cells, disappears upon treatment with the alkaline phosphatatse. These results suggest that phosphorylation of TBP may play a role in down-regulation of SLl activity. In addition, we attempted to analyze the phosphorylation state of SLl before and after differentiation of U937 cells using in vivo labeling experiments. Unfortunately, these experiments were inconclusive because of the inability to detect SLl, an extremely low abundance factor in the cell, in immunoprecipitates from [3 2 P]Pi-labeled cell extracts. In addition to determine the activity of SLl, we have also analyzed the transcriptional properties of the partially purified UBF fractions. The results of the in vitro reconstituted transcription assays indicate that UBF activity in differentiated cells is higher than that in undifferentiated cells. The significance of these findings 141 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. is currently unclear and requires further investigation. Nevertheless, these results further support our finding that sown-regulation of SLl is at the basis of pol I repression upon U937 cell differentiation. Previous studies indicated that differentiation of U937 is accompanied by a relocalization of pRB to the nucleolus (Cavanaugh et al., 1995; Rogalsky et al., 1993). Once in the nucleolus, pRb can bind to UBF (Cavanaugh et al., 1995; Voit et al., 1997). These findings led to the demonstration that, in vitro, pRb can repress pol I transcription by directly binding to UBF (Rogalsky et al., 1993; Voit et al., 1997). However, it has never been shown that the activity of UBF is affected upon U937 cell differentiation. On the other hand, our biochemical studies strongly suggest that sown-regulation of SLl activity is at the basis of the repression of pol I transcription in differentiated U937 cells. In addition, our data show that UBF is more active in the differentiated cells. The difference between our findings and the published data may reflect difference in the experimental approach. It is possible that in the reconstituted transcription assays using mouse extracts, recombinant pRb is sufficient for the inhibition of pol I transcription by binding, stoichiometrically, to UBF. However, it is unclear if there is a stoichiometric interaction between pRb and UBF in differentiated U937 cells. Conversely, our analysis has been performed on endogenous factors using U937 cell extracts; therefore, it may better recapitulate the process that occurs in vivo. Our study can not rule out that the binding of pRb to UBF is still required for repression of pol I transcription in differentiated U937 cells. It is conceivable that the interaction between UBF and pRb may represent one step in 142 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. a process that ultimately leads to the down-regulation of SLl activity and repression of pol I transcription. In analogy to the recently proposed mechanism of pRb repression of class II genes, pRb may function in the recruitment of other factors to the rDNA promoter that facilitate or directly catalyze the inactivation of SLl (Brehm et al., 1999). In conclusion, our study underscores the role of SLl as a critical target for regulation of pol I transcription in differentiated hematopoietic cells. The precise role of pRb in this process remains to be elucidated, and future studies will address the link between SLl, pRb and repression of pol I transcription in differentiated hematopoietic cells. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. APPENDEX B Molecular Mechanism of Down-Regulation of NF-kB by IL-10 144 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.l Background Interleukin 10 (IL-10) is a pleiotropic cytokine produced by ThO and Th2 T cells, CD5+ B cells, thymocytes, kerotinocytes and monocytes/microphages (Moore et al. 1990; Moore et al. 1993). IL-10 is a major endogenous anti-inflammatory mediator. It suppresses the production of macrophage inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNFa at their transcription levels, and it can suppress T cell responses by downregulating the expression of MHC class II molecules and costimulatory molecules such as ICAM-1 and B7 (Fiorentino et al. 1991; Bogdan et al. 1992; Bogdan and Nathan 1993; Wang et al. 1994; Willems et al. 1994; Bogdan et al. 1991; de Waal Malefyt et al. 1991). In addition, it can diminish the Thl cell activity by the suppression of IL-2 and interferon-y (Moore et al., 1993). In vivo studies support the anti-inflammatory and immunosuppressive function of IL-10. IL-10 deficient mice develop chronic enterocolitis with similarities to inflammatory bowel disease (Kuhn et al., 1993). IL-10 treatment has been shown to improve colitis (Powrie et al., 1993; Herfarth et al., 1996; Herfarth et al., 1998) and arthritis (Herfarth et al., 1996; Walmsley et al., 1996), autoimmune encaphalomyelitis, pancreatitis, diabetes mellitus and endotoxemia in animal models (Gerard et al., 1993; Pennline et al., 1994; Rott et al., 1994; Van Laethem et al., 1995). Moreover, IL-10 has been shown to improve patients suffering from Crohn’s disease and Rheumatoid Arthritis in clinical trials (Schreiber et al., 1995; van Deventer et al., 1997). It is important to realize that IL-10 also has immunostimulatory effects to B cell proliferation and differentiation (Go et al., 1990; Defrance et al., 1992; Rousset 145 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. et al., 1992). In summary, IL-10 is a major regulatory cytokine that suppresses Thl dependent cell mediated immune response, while supporting Th2 dependent humoral response. IL-10 exerts its biological functions by interacting with its cell surface receptors. The functional active IL-10 receptors are composed of two subunits: IL-10R1 and IL-10 R2. Both of them belong to the class II cytokine receptor family. IL-10R1 is a 110 kDa glycoprotein primarily expressed by hematopoietic cells such as B cells, T cells, NK cells, monocytes and macrophages (Donnelly et al., 1999). IL-10R1 plays the dominant role in mediating high affinity ligand binding and in signal transduction (Ho et al., 1993). IL-10R2 is a 40kDa polypeptide expressed by most tissues (Gibbs et al., 1997; Kotenko et al., 1997). IL-10R2 is an essential component of functional IL-10R complex and is largely required only for signaling (Spencer et al., 1998). IL-10R2 knockout mice develop chronic colitis which underscores the critical role of IL-10 as a negative regulator of inflammation (Spencer et al., 1998). Forced expression of the IL-10R1 chain is sufficient for many cell types to be able to respond functionally to IL-10, however, in some cell types this is not sufficient (Riley et al., 1999). Upon the binding of IL-10, the IL-10 receptors aggregate and activate the JAK-STAT signaling pathway. IL-10 effects the activation of Jakl, which is associated with the cytoplasmic domain of IL-10R1, and Tyk2, which is associated with the IL-10R2 (Finbloom et al., 1995; Ho et al., 1995; Wehinger et al., 1996). The Jaks transphosphorylate tyrosine residues on IL-10R1 (Tyr-427 and Tyr-477 in 146 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mouse, and Tyr-446 and Tyr-496 in human) (Riley et al., 1999). The phosphorylated tyrosine residues then serve as docking sites for the cognate Statl , Stat3 and in some cells Stat5 (Finbloom et al., 1995; Ho et al., 1995; Wehinger et al., 1996). The Stat proteins are recruited to the receptors and get phosphorylated by receptor associated Jaks. The phosphorylated Stats are then released from the docking site and form homodimers and/or heterodimers. They translocate to the nucleus and bind to the regulatory DNA elements as transcription factors. It has been reported that Jakl and Stat3 are required for IL-10 to inhibit TNFa production in LPS-stimulated macrophages (Meraz et al., 1996; Rodig et al., 1998; Riley et al., 1999). The two tyrosine residues, Tyr-427 and Tyr-477, in the mouse IL-10R1 and the 30 amino acids at the carboxyl terminal sequence containing at least one functionally critical serines are required for IL-10's anti-inflammatory function (Riley et al., 1999). IL-10 can inhibit LPS induced monocytes activation by blocking the Ras signaling pathway (O'Farrell et al., 1998). IL-10 has also been shown to interfere with protein-tyrosine kinase-dependent CD40 signaling controlling IL-ip synthesis in monocytes (Weber-Nordt et al., 1994). Many of the proinflammatory cytokines and co-stimulatory proteins which are suppressed by IL-10 are reported to be regulated by the transcriptional factor NF-kB. NF-kB is a eukaryotic transcription factor that exists in virtually all cell types. NF-kB is the key regulator of the expression of numerous cytokines and adhesion molecules involved in inflammatory responses (Ghosh et al., 1998). The prolonged activation of TNF and IL-1, which are the target genes of NF-kB, is the 147 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. major cause in chronic inflammatory diseases such as rheumatoid arthritis, asthma and psoriasis (Barnes et al., 1997; Ghosh et al., 1998). N F - k B is composed of a family of proteins, and the classical N F - kB refers to a heterodimer composed of p65 and p50 (Finco et al., 1995). In most cells, N F - k B is retained in the cytoplasm in an inactive form by binding to the inhibitor, IkB o, (Finco et al., 1995), which is also composed of a family of proteins. In response to a variety of extracellular stimuli, such as IL-1 (3 , TNFa, LPS, etc., IkB are rapidly phosphorylated by Ik B kinase (IKK) complex. The IKK complex is composed of IKK-a, IKK-p and IKK-y (Rothwarf et al., 1998). The IKK complex then phosphorylates the two serine residues (ser32 and ser36) of IkB o c (Brown et al., 1995; DiDonato et al., 1997). The phosphorylated IkB o, will be ubiquitinated and targeted to the 26s proteasome (Finco et al., 1994). The degradation of Ik B o c results in the release of N F - kB , which then translocates to the nucleus and transactivates different genes. These genes include IL-1, IL-6, IL-8, TNFa, GM-CSF (Ghosh et al., 1998). IL-10 has been found to inhibit the activity of NF-kB in monocytes/macrophages and T cells (Wang et al., 1995; Dokter et al., 1996; Romano et al., 1996; Lentsch et al., 1997; Clarke et al., 1998). Two groups reported that the DNA binding activity of NF-kB was not inhibited by the IL-10 treatment (Dokter et al., 1996; Clarke et al., 1998). However, there are other six groups described the inhibition of NF-kB DNA binding activity by IL-10 (Romano et al., 1996; Riley et 148 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. al., 1999; Koenig et al., 2001). There has been three groups reported that the suppression of DNA binding activity of NF-kB is resulted from the inhibition of IkB degradation (Lentsch et al., 1997; Shames et al., 1998; Clarke et al., 1998). Nine months after we initiated this project, it was determined that the IKK activity is inhibited by IL-10, which results in the suppression of NF-kB DNA binding activity and the inhibited degradation of IkB (Schottelius et al., 1999). Our hypothesis is that since NF-kB is the key factor mediating pro-inflammatory responses, while IL-10 is the prime anti-inflammatory regulator, IL-10 could downregulate NF-kB by inhibiting the activation of IKK, or by inhibiting the degradation of phosphorylated IkB. The goals of our studies are: 1) to identify at which level of the NF-kB pathway the IL-10 downregulates the activation of NF-kB. Is it at the level of IKK or at the degradation of phosphorylated IkB? This study will help us to understand the molecular mechanism of IL-10 downregulation of NF-kB. 2) to determine the functional domain of IL-10R1 involved in downregulating NF-kB activation. This will help us to determine the down stream molecules in the IL-10R1 pathway which are involved in the downregulation of NF-kB. Eventually, we can define the converging point of IL-10 and NF-kB signaling pathways. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B .2 A b s tr a c t Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by nuclear factor kB (N F - kB ). In its inactive state, N F - kB is retained in the cytoplasm by bounding to inhibitor IkB . Upon activation, the degradation of IkB enables N F -kB to be released and translocate into nucleus activating gene transcriptions. Conflicting data exists on the effects of IL-10 on TNF induced N F - k B activity in human monocytes and the molecular mechanism involved remains to be elucidated. In this study, we show that IL-10 functions to inhibit degradation of IkB and the DNA binding activity of N F - kB . Our results indicate that the last 28 amino acids at the carboxyl terminal of IL-10 receptor 1 (IL-10R1) are required for downregulation of N F - k B . Moreover, Janus tyrosine kinases Jakl and Tyk2 are not involved in the downregulation of N F - kB by IL-10. The finding of the requirement of the C-terminal 28 amino acids of IL-10R1 will assume greater importance in discovering potential novel mediators in downregulating N F - kB by IL-10. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.3 Introduction Interleukin 10 (IL-10) is a pleiotropic cytokine produced by ThO and Th2 T cells, CD5+ B cells, thymocytes, kerotinocytes and monocytes/microphages (Moore et al. 1990; Moore et al. 1993). IL-10 conducts its anti-inflammatory effect by suppressing production of macrophage inflammatory proteins such as IL-1, IL-6, IL-8, IL-12, TNF, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, MHC class II molecules, B7, and intercellular adhesion molecule-1 (Bogdan and Nathan, 1993; Fiorentino et al., 1991; de Wall Malefyt et al., 1991; Bogdan et al., 1991; Wang et al., 1994a; Wang et al., 1994b; Bogdan et al., 1992; Willems et al., 1994) and through diminishing Thl cell activity by suppression of IL-2 and interferon-y (Moore et al., 1993). IL-10 has been shown to be effective in preventing or inhibiting inflammation and autoreactivity in vivo in a variety of rodent models. IL-10 has therapeutic effects in several models of autoimmunity, including the prevention of diabetes in nonobese diabetic mice (Pennline et al., 1994), beneficial effects in experimental allergic encephalitis in rats (Rott et al., 1994) as well as protection of mice from experimental allergic thyroiditis (Mignon-Codefroy et al., 1995). Moreover, treatment with IL-10 ameliorates established arthritis in mice (Joosten et al., 1997; Persson et al., 1996; Walmsley et al., 1995). In consistence, IL10 knockout mice develop chronic inflammatory bowel disease and contact hypersensitivity response (Rennick et al., 1995). Preliminary results from clinical studies have shown improvement in Rheumatoid Arthritis 151 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. (Maini et al., 1997) and Crohn’s disease patients (Schreiber et al., 1995; van Deventer et al., 1997). Many of the proinflammatory cytokines and co-stimulatory proteins which are suppressed by IL-10 are reported to be regulated by the transcriptional factor N F - kB (Baldwin, 1996). N F -k B is a ubiquitously expressed and regulates the induction of genes involved in immune and inflammatory cell function (Verma et al., 1995; Baldwin, 1996-3), and in anti-apoptotic responses (Wang et al., 1996). N F - kB is composed of homo- and heterodimers of Rel family members, typically p65 and p50 which are retained in the cytoplasm by binding to inhibitor protein Ik B (Finco and Baldwin, 1995). Upon the activation by a variety of stimuli, such as IL-1 (3 , TNF, LPS, or phorbol esters, IkB is phosphorylated on two conserved serine residues near its N-terminus, which targets it for ubiquitylation and degradation by the 26S proteasome (Baldwin, 1996; DiDonato et al., 1996; Brown et al., 1995; Finco et al., 1994). The liberated N F - kB subsequently translocates into nucleus where it binds to cognate sites and stimulates transcription of traget genes. The phosphorylation of IkB is carried out by iKB-kinase complex (IKK complex), which is composed of IKK-a, IKK-[ 3 and IKK-y/NEMO (Rothwarf et al., 1998). IKK-a and IKK-J3 share significant sequence homology; however, they have different functions whereby IKKa is essential for limb development and skin differentiation (Takeda et al., 1999; Hu et al., 1999; Li et al., 1999), and IKKP is important for induction of N F - kB by cytokines. 152 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Although IL-10 has been found to inhibit the activity of N F - kB in monocytes/macrophages and T cells, there has been conflicting data as to whether IL-10 inhibits DNA binding activity of N F - kB (Wang et al., 1995; Dokter et al., 1996; Romano et al., 1996; Clarke et al., 1998; Lentsch et al., 1997). Inhibition of IkB degradation has also been reported as a mechanism of downregulation of N F - kB by IL-10 (Lentsch et al., 1997; Shames et al., 1998; Clarke et al., 1998). In this study, we intend to clarify this question by determining the step in the N F - kB activation pathway that is inhibited. Further more, we would like to define the domains of IL-10R1 which are required for the downregulation of N F - k B . Our results indicate N F - kB DNA binding activity is inhibited by IL-10 in murine RAW 264.7 cells. In addition, IkB degradation is inhibited by IL-10 in human HL-60 T cells. We also demonstrated that Janus kinases, Jakl and Tyk2, which are activated by IL-10 in the JAK-STAT signaling pathway, are not involved in downregulating N F - kB by IL-10. Additionally, the deletion mutagenesis studies of IL-10R1 revealed that the 28 amino acids at the carboxyl terminus are required for the integral function of IL-10 in inhibiting N F - kB activation. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.4 Material and Methods C e ll C u ltu r e a n d T r e a tm e n t: Human monocytic cell line U937 and human HL-60 T cell line were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS. Mouse Macrophage Raw264.7 cells, Hela S3 cells, U1 (TykT) and U4 (JaklT) cells were cultured in DMEM supplemented with 10% heat- inactivated FCS. All cell lines were incubated at 37°C, 5% CO2 in humidified atmosphere. Cells were grown to a density of 1 X 106 /ml and stimulated with 20 ng/ml human or murine TNFa, in the presence or absence of 10 ng/ml pretreatment of human or murine IL-10 for various time periods. Cells were then harvested and processed for electrophoretic mobility shift assays and Western blot analysis. E le c tr o p h o r e tic M o b ility S h ift A ssa y s (E M S A ): Nuclear extracts were prepared as described in DiDonato et al., 1996. Briefly, the cells were harvested in ice-cold phosphate-buffered saline (PBS) and pelleted at 2,000g at room temperature for 30 seconds. The cell pellet was resuspended in buffer A[10 mM HEPES (pH7.9), lOmM KC1, 0.1 mM EDTA, 0.1 mM EGTA and 2.5 pM DTT, ImM PMSF and 5 pg/ml leupeptin] and incubated on ice for 15 mins. Then 0.5% NP-40 was added and the cell lysate was centrifuged down at 13,000g for 5 mins at 4 °C. The pellet was resuspended in IX lysis buffer as described previously, with 0.5% NP-40, and 400 mM KC1. After incubation on ice for 20 mins, lysate was centrifuged at 13,000g for 5 mins at 4 °C. The supernatant is nuclear extract. 10 mg of nuclear extract was incubated with 5 mg of polyoligonucleotides (dl-dC) and 20,000 cpm (0.2 ng) of the 154 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3 2 P labeled oligonucleotide probe corresponding to the NF-KB-binding site (5’-AGTTGAGGGGACTTTCCCAGGC-3 ’). After 30 min of incubation on ice, the bound and free DNAs were fractionated on 4% native polyacrylamide gels. Dried gels were exposed to Phosphorlmager (Molecular Dynamics). RT-PCR of IL-10R1 and IL-10R2: mRNA from various cell lines were extracted using mRNA isolation kit following the manufacturer’s instructions (Amersham Pharmacia). Briefly, the first strand cDNA was generated using random hexmer and M-MLV reverse transcriptase. DNA fragments of IL-10R1 and IL-10R2 were generated by PCR using specific primers. For IL-10R1, primers are 5’-GTC TGA AAG TAC CTG CTA TGA AG-3’ and 5’-GAG GAT GAA GCC ATT GTG GAT S’ which generate a 420-bp fragment. For IL-10R2, the specific primers are 5’-GAG TCA CCT GCT TTT GCC AAA-3’ and 5’-CCA GTC TGA ATG CTC ATC TGC-3’ which generate a 180-bp fragment. Cloning and mutagenesis of human IL-10R1: Vector pEF3-IL-10Rl, which contains wild type human IL-10R1, was obtained from Serguei Kotenko (Kotenko et al., 1997). To construct plasmid pUHD10-3-IL-10Rl, which is the response plasmid in the tet-on inducible system, IL-10R1 was subcloned by digesting pEF3-IL-10Rl with EcoRl and Spel and ligated to EcoRI and Xbal digested vector pUHD10-3 (Resnitzlcy et al., 1994-5). To construct plasmid encoding double-point mutation where tyrosines at position 446 and 496 were mutated to phenylalanine, single-point 155 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mutation at position 446 was used as the template for the second mutation as position 496. Mutation at position 446 was established by PCR using primers that contain the point mutation. The two primers were 5’-TTC CAG GGT TTC CTG AGG CAG-3’ and 5’-CTG CCT CAG GAA ACC CTG GAA-3’. The primers were first phosphorylated before being used in PCR reactions. The PCR products were digested with Dpnl to cleave wild type methylated templates. The digested PCR products were transformed to DH5a competent cells and single clones that contain the single point mutation at position 446 were selected and sequenced. To make the double mutation plasmid pUHD10-3-IL-10Rl-FF, single point mutation at 446 was used as the template and the primers were 5’-GCC AAG GGC TTT TTG AAA CAG-3’ and 5’-CTG TTT CAA AAA GCC CTT GGC-3’. Plasmid pUHD10-3-IL-10Rl-A28, which encodes a deletion mutation lacking the last amino acids at the carboxyl terminal, was generated by direct linker-PCR cloning by using wild type IL-10R1 as template and primers 5’-CCC GAA TTC TGC AGA TCG CGC CCA-3’ and 5’-CCC ACT AGT TCA CAC ACA GCC TGA-3’. Plasmid pUF!D10-3-IL-10Rl-FF/A28 which carries double-point mutation and deletion mutation was generated by linker-PCR cloning using pUHD10-3-IL-10Rl-A28 as template and the same primers used for generating deletion mutation. Plasmid pUF[D10-3-IL-10Rl-Ser/Ala encodes a mutation where the last sic serines in the carboxyl terminal of IL-10R1 were mutated to alanines. This plasmid was generated by three-piece ligation where the mutations were contained in synthesized double-stranded DNA oligomer. 156 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Transient transfection and induction of expression: Hela cells were transiently co-transfected with response plasmid which carries wild type or mutant IL-10R1 in vector pUHD10-3 and with plasmid pUHD172-lneo (Gossen et al., 1995) which encodes for rtTA gene for the Tet-on system. DNA amount ratio for pUHD10-3 based plasmids to pUHD172-lneo was 10:1. Transfection was conducted following manufacturer’s instructions (LipofectAmine Plus, Invitrogene). 24 hrs post-transfection, 1 pg/ml doxycycline (DOX) was added to the media to induce the expression of wild type or mutant IL-10R1. W e s te r n B lo t A n a ly sis: Whole cell extracts were prepared as described in DiDonato et al.(1996). Briefly, Briefly, the cells were harvested in ice-cold phosphate-buffered saline (PBS) and pelleted at 2,000g at room temperature for 30 seconds. The pellet was resuspended in lysis buffer [0.1% NP-40, 50 mM Tris-HCl (pH8), 0.15 mM NaCl with cocktail of proteinase and phosphotase inhibitors]. Protein concentrations were determined by Bradford assay. Equal amounts of extracts were subjected to SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. The degradation of I k B a was detected by polyclonal antibodies specific to Ik B c x , followed by horseradish peroxidase-linked anti-rabbit IgG antibodies and detected by chemiluminescence. I m m u n o flu o r e s c e n c e S ta in in g : Hela cells, U 1 cells and U4 cells were cultured on cover glasses in 6-well plates with 1 X 106 cells/well. Cells were washed with PBS 157 R eproduced with perm ission o f the copyright owner. Further reproduction prohibited without perm ission. and then fixed with 3.7% formadehyde for 15 min at room temperature. After washing with wash buffer (1% triton in PBS), specimens were incubated with blocking solution (1% triton, 1% non-fat milk and 1% BSA in PBS) for 30 min at room temperature. Then the specimens were incubated with rabbit-anti-IL-lORl antibodies and mouse-anti-lKBa antibodies. After three times 5-min washes with wash buffer, secondary antibodies conjugated with FITC or rhodamine were then applied. Then the nucleus of specimens were stained by Hoechest (0.1 pg/ml) for 20 min at room temperature, after washing 3X with washing buffer and 2X with PBS. After wash 3 X with PBS, the specimens were subjected to fluorescent microscope. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5 Results B .5 .1 N F - k B D N A -B in d in g A c tiv ity is In h ib ite d b y IL -1 0 in H L -6 0 T C e lls There has been inconsistent data regarding the inhibition of NF-kB DNA binding activity upon the treatment of IL-10. Two groups reported that the DNA binding activity of NF-kB was not inhibited by the IL-10 treatment (Dokter et al., 1996; Clarke et al., 1998), while the other six groups described the inhibition of NF-kB DNA binding activity by IL-10 (Romano et al. 1996; Riley, Lentsch et al., 1997; Shames et al., 1998; Takeda et al. 1999; Schottelius et al., 1999; Sica et al. 2000). To clarify this question, different lymphoid human cell lines (HL-60 T cells and U937 monocyte cells) and murine cell lines (macrophage RAW264.7) were used for this study. Nuclear extracts were prepared from cells treated with TNFa with or without pretreatment of IL-10. The NF-kB DNA binding activities to consensus site were detected by EMSA. Analysis of nuclear extract from TNF stimulated RAW264.7 cells demonstrated an increase in NF-kB DNA binding activity as compared with nuclear extracts from unstimulated cells and cells treated with IL-10 alone (Fig. B-l, lane 5 and lanes 1-4, respectively). Pretreatment with IL-10 inhibited TNF induced DNA binding in a dose-dependent manner (Fig, B-l, lanes 6-8). Similar results were obtained using U937 cells and HL-60 T cells (data not shown). Therefore, these results are consistent with the reports that IL-10 treatment can inhibit DNA-binding activity of NF-kB. 159 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5.2 IL-10 Inhibits NF-kB DNA-Binding Activity by Preventing TNFa I n d u c e d D e g r a d a tio n o f L cB a Since NF-kB is retained in the cytoplasm by binding to the inhibitor IkB, one possible mechanism for IL-10 to inhibit NF-kB DN-binding activity is through inhibiting phosphorylated IkB degradation upon TNF induction. To address this question, HL-60 T cells were stimulated by TNFa for 20 mins in the presence or absence of pretreatment of IL-10. Whole cell extracts were used for Western blot analysis blotted against iKBa protein. As shown in figure B-2, degradation of iKBa was inhibited by IL-10 in a dose-dependent manner (lane 6-8). These results indicate that IL-10 inhibits NF-kB DNA-binding activity by preventing TNFa-induced degradation of IKBa, which consequently preventing nuclear translocation of NF-kB. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TNFa _ - _ _ 20 20 20 20 ,IL',10n 0 10 50 100 0 10 50 100 (n g /m l) t o w w i * . « *- nf-kb non-specific Figure B-l. NF-kB DNA-binding activity is inhibited by IL-10 in RAW264.7 cells. Murine Raw264.7 cells were pretreated with or without murine IL-10 at various concentrations as indicated for 15 mins, followed by treatment with TNFa (20 ng/ml) for 20 mins. Nuclear extracts were obtained and were incubated with 32P labeled oligonucleotide probe corresponding to the NF-kB binding site. The DNA-protein complexes were analyzed by EMSA as described. - - - + + + + 2 10 20 0 2 10 20 TNF (20 ng/ml) IL-10 (ng/ml) — IicBa Figure B-2. IL-10 inhibits NF-kB DNA-binding activity by preventing TNFa induced degradation of IicBa. HL-60 cells were pretreated with various amount of human IL-10 for 15 mins as indicated, followed by TNFa treatment for 20 mins. Equal amount of whole cell extracts were resolved on 10% SDS-PAGE, and blotted with anti-bodies specific to IxBa. 161 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5.3 IL-10 Inhibits NF-kB DNA-Binding Activity through Inhibiting TNF-Induced Translocation of I k B To further prove that inhibition of NF-kB DNA-binding activity by IL-10 is caused by inhibition of degradation of hcBa, and consequently preventing nuclear translocation of NF-kB, we employed immunostaining assays which enable direct visualization of NF-kB translocation. To clearly demonstrate that IL-10 inhibits NF-kB DNA-binding activity through preventing its nuclear translocation, inducible expression of IL-10R1 were obtained using tet-on system in Hela cells. Hela cells constitutively express IL-10R2, which is an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events (Kotenko et al., 1997). As shown in figure B-3, expression of IL-10R2 expression was detected by RT-PCR with primers specific to IL-10R2 (lane 8), whereas expression of IL-10R1 in Hela cells was not detected (lane 2). To express IL-10R1 in the tet-on inducible system, plasmid pUHD10-3-IL-10Rl, which contains wild type IL-10R1, and pUHD172-lneo, which encodes transcription activator rtTA, were contransfected to Hela cells. 24 hours post-transfection, doxycycline was added to the media to induce the expression of IL-10R1 for 7 hours. Thus, tightly controlled expression of IL-10R1 can avoid potential adverse effects of constitutively expression in Hela cells that do not express IL-10R1 endogenously. As shown in figure B-4, treatment of TNFa triggered translation of p65, a component of NF-kB complex, into the nucleus. In contrast, pre-treatment of IL-10 abolished nuclear translocation of p65. However, some cells treated with TNFa only also showed 162 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. blockage of NF-kB nuclear translocation. This could be due to highly expressed IL-10R1 somehow physically interfered TNF receptor trimerization. These results strongly support previous results that IL-10 can inhibit NF-kB DNA-binding activity as demonstrated in EMSA assays. The inhibition o f IkB degradation could be caused by two mechanisms: 1) this could be caused by inhibited ubiquitination of phosphorylated IkB, therefore the subsequent targeted for 26S proteasome degradation; 2) alternatively, this could be caused by inhibiting activation of IKK, which phosphorylates IkB. To determine the mechanism, IKK activities were measured in TNFa treated cells in the presence or absence of IL-10 in primary cells and in cell lines. Unfortunately, the results were not conclusive. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. E x p r e s s i o n o f I L - 1 0 R 1 / R 2 I n D iffe re n t C ell L in e s ( R T - P C R ) M 123456123456 < - IL-10R1 < ~ I.L-10R2 1. N eg . con trl 4. HL-60 — ► 2. H ela S. Jurkat 3, Tet-H ela ©, PBMG Figure B-3. RT-PCR analysis of IL-lORl and IL-10R2 expression on different cell lines. mRNA from different cell lines were extracted and reverse transcribed to first strand cDNAs. DNA fragments were generated by PCR using specific primers. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. p©S I L - I O R I D A R I Now- Treated TNF IL-10 TNF n J r o Figure B-4. IL-10 inhibits NF-kB DNA-binding activity through inhibiting TNFa induced translocation of iKBa. Hela cells were transiently transfected with pUHD10-3-IL-10Rl, which encodes wild type IL-10R1 and pUHD172-lneo, which encodes transcription activator rtTA. 24 hr post-transfection, DOX (lpg/ml) were added to induce IL-10R1 expression for 7 hr. Cells were then treated with or without TNFa in the presence or absence of IL-10, subjected to immunostaining analysis with anti-p65 antibodies (red), anti-IL-lORl antibodies (green) as described in Material and Methods. The nucleus of specimens were stained by Hoechest 33258 (blue). 165 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5.4 Tyk2 and Jakl are Not Required to Inhibit TNF-induced IkB Translocation by IL-10 IL-10 exerts its biological function through the activation of Jakl and Tyk2, the members of the receptor-associated Janus tyrosine kinase (Jalc) family (Dusanter-Fourt et al, 1994; Ihle et al., 1995). Upon IL-10 induction, IL-10R1 is associated with Jakl and I1-10R2 is associated with Tyk2. The binding of the ligand homodimers to the R1 chains results in formation of a non-functional intracellular receptor complex (Kotenko et al., 1997). The second chain R2 are required to assemble an active intracellular receptor complex and thus to initiate the signal transduction cascade. Ligand induced oligomerization of receptor components results in Jakl and Tyk2 activation and the subsequent phosphorylation of tyrosine 446 and 496 residues of IL-10R1. These phosphorylated tyrosine residues and their flanking peptides serve as docking sites for latent cytosolic transcription factors Statl, Stat3 and Stat5 (Finbloom and Winestock, 1995; Ho et al., 1995; Weber-Nordt et al., 1996). The Stats are then phosphorylated on Tyr residues by the receptor associated kinases, resulting in homo- or heterodimers. These dimers subsequently dissociate from receptors and translocate to nucleus. However, IL-10R2 intracellular domain does not contain Stat docking sites and thus do not participate in the Stat recruitment process. U4 and U1 are Jakl and Tyk2 negative cells. To determine whether Jakl or Tyk2 is required for preventing NF-kB translocation upon IL-10 stimulation, U4 and U1 cells were transiently co-transfected with pUHD10-3-IL-10Rl, which contains wild type IL-10R1, and pUHD172-lneo, which 166 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. encodes transcription activator rtTA. The transfected cells were inducted by DOX for 7 hours 24 hours post-transfection and were subjected to immunostaining as described previously. The blockages of p65 translocation upon IL-10 treatment were observed in both U4 (Fig. B-5) and U1 cells (Fig.B-6). This indicates that Jakl and Tyk2 are not likely to be involved in inhibiting NF-kB translocation by IL-10 treatment. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. p 6 5 IL-10 R 1 D A P I N o n - T rea ted T N F TN F IL -10 Figure B-5. Jakl is not required to inhibit TNFa-induced iKBa translocation by IL-10 in U4 (Jakl'/') cells. Cells were treated with or without TNFa in the presence or absence of IL-10, followed by immunostaining analysis with anti-p65 antibodies (red), anti-IL-lORl antibodies (green) as described in Material and Methods. The nucleus of specimens were stained by Hoechest 33258 (blue). 168 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. p6S IIL-1OR1 DAP! Non- T re a te d : T N F T N F l l J l O Figure B-6. Tyk2 is not required to inhibit TNFa-induced iKBa translocation by IL-10 in Ul (Tyk2T) cells. Cells were treated with or without TNFa in the presence or absence of IL-10, followed by immunostaining analysis with anti-p65 antibodies (red), anti-IL-lORl antibodies (green) as described in Material and Methods. The nucleuses of specimens were stained by Hoechest 33258 (blue). 169 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5.5 Tyrosines at positions 446 and 496 of IL-10R1 at the Stat Docking Site Are Not Required to Inhibit TNF-induced IkB Translocation by IL-10 To illustrate the molecular mechanism of IL-10 inhibiting N F - kB activation, it is necessary to determine the functional domains of IL-10R1. It is known that Tyr-427 and Tyr-447 of murine IL-10R1, which are equivalent sites of human Tyr-446 and Tyr-496, are required for activating Jak-Stat pathway (Weber-Nordt et al., 1994). It has also been demonstrated that the ability of IL-10 to inhibit TNFa production in LPS stimulated macrophages requires the presence of Stat3 and Jakl (Riley et al., 1999). We therefore would like to determine whether these two tyrosine residues are required for inhibiting N F - kB activation by IL-10. To study this, the two tyrosines on the Stat docking site on IL-10R1 were mutated to phenylalanines. Hela cells were transiently co-transfected with pUHD10-3-IL-10Rl-FF, which contains mutated IL-10R1, and pUHD172-lneo, which encodes transcription activator rtTA. The transfected cells were inducted by DOX for 7 hours 24 hours post-transfection and were subjected to immunostaining as described previously. It is observed that the cells expressing phenylalanine mutation can still prevent p65 from translocating to nucleus compared with wild type IL-10R1 as shown in figure B-7. Therefore, the results suggest the Stat3 docking sites on IL-10R1 are not required in inhibiting N F - k B activation by IL-10. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. P 6 5 1 L .-1 0 R 1 D A P t Nort- Trdated T N F Figure B-7. Tyrosines at positions 446 and 496 of IL-10R1 at the Stat docking site are not required to inhibit TNFa-induced IicBa translocation by IL-10. Hela cells were transiently transfected with pUHD10-3-IL-10Rl-FF, which encodes two tyrosine-to-phenylalanine point mutations at positions 446 and 496 on IL-10R1 and pUHD172-lneo, which encodes transcription activator rtTA. 24 hr post-transfection, DOX (Ipg/ml) were added to induce IL-10R1 expression for 7 hr. Cells were then treated with or without TNFa in the presence or absence of IL-10, subjected to immunostaining analysis with anti-p65 antibodies (red), anti-IL-lORl antibodies (green) as described in Material and Methods. The nucleuses of specimens were stained by Hoechest 33258 (blue). 171 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.5.6 28 Amino Acids at the Carboxyl Terminal of IL-10R1 are Required to Inhibit TNF-induced IkB Translocation by IL-10 It has been reported that the anti-inflammatory function of IL-10 requires the last 30 amino acids at the carboxyl terminal sequence of the murine IL-10R1 in the intracellular domain (Riley et al., 1999). More importantly, the mutation of all four serine residues in the carboxyl terminal of IL-10R1 failed to inhibit LPS-mediated TNFa production in murine macrophages (Riley et al., 1999). Since LPS is a potent activator of NF-kB, we are interested in determining whether the carboxyl terminal of human IL-10R1 is also required in the inhibition of TNFa stimulation of NF-kB. To study this, plasmid pUHD10-3-IL-10Rl-A28, which encodes a deletion mutation lacking the last amino acids at the carboxyl terminal, was generated. This plasmid was co-transfected with pUHD172-lneo, which encodes transcription activator rtTA, to Hela cells. 24 hours post-transfection, the expression of the carboxyl terminal truncated IL-10R1 was induced by adding DOX for 7 hours. The cells were then treated with TNFa in the presence or absence of IL-10. It is observed that the truncated IL-10R1 fails to block p65 translocation to the nucleus (Fig. B-8). This result indicates that the carboxyl terminal of human IL-10R1 is required in downregulating NF-kB activation by inhibiting the translocation of p65. However, the blockage of p65 translocation can also be detected in cells over-expressing IL-10R1 deletion mutant independent of IL-10 treatment (panel 2 of fig. B-3). One of the explanations for this observation is that the overexpressed IL-10R1 on the cell membrane can overthrow the TNF pathway by physically blocking the aggregation 172 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. of TNF receptors upon stimulation. To pin-point whether the 6 serine residues in the carboxyl terminal are required for this function, all last six serines in the sequence were mutated to alanines. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. P® » IL.-IORI D A P I N on- TN F TN F I L -1 0 Figure B-8. The 28 amino acids at the carboxyl terminal of IL-10R1 are required to inhibit TNFa-induced IkBoc translocation by IL-10. Hela cells were transiently transfected with pUHD10-3-IL-10Rl-A28, which encodes IL-10R1 with 28 amino acid deletion at C-terminal and pUHD172-lneo, which encodes transcription activator rtTA. 24 hr post-transfection, DOX (lpg/ml) were added to induce IL-10R1 expression for 7 hr. Cells were then treated with or without TNFa in the presence or absence of IL-10, subjected to immunostaining analysis with anti-p65 antibodies (red), anti-IL-lORl antibodies (green) as described in Material and Methods. The nucleus of specimens were stained by Hoechest 33258 (blue). 174 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. B.6 Discussion Interleukin-10 is a pleiotropic cytokine that inhibits cell-mediated immune responses while enhancing humoral immunity (Moore et al., 2001). IL-10 controls inflammatory processes by suppressing the production of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNFa, IL-12 and interferon-y in monocytes, macrophages, T cells and other various cell types (de Wall Malefyt et al., 1991; Fiorentino et al., 1991; Bodgan et al., 1992; Wang et al., 1994a; Wang et al., 1994b). This inhibition of cytokine production is accompanied by reduced mRNA productions (de Wall Malefyt et al., 1991; Fiorentino et al., 1991; Bodgan et al., 1992; Wang et al., 1994a; Wang et al., 1994b). Gene promoters for these proinflammatory cytokines contains cis-acting motifs for transcription factor N F - k B which has been indicated to be critical for induction of these cytokines (Shirakawa et al., 1993). Although IL-10 has been found to inhibit the activity of N F - kB in various reports, there is no consensus conclusion as to whether the DNA binding activity of N F - kB is inhibited. Two groups reported that the DNA binding activity of N F - kB was not inhibited by the IL-10 treatment (Dokter et alk, 1996 and clarke et al., 1998), while the other six groups demonstrated otherwise (Romano et al., 1996; Riley et al., 1999, Shames et al., 1998; Takeda et al., 1999; Schottelius et al., 1999 and Sica et al., 2000). Here we demonstrated that N F - kB DNA-binding activity is inhibited by IL-10 in HL-60 T Cells (Fig. B-l) and monocytic U937 cells (data not shown). This finding is further supported by the observation that IL-10 pretreatment was able to suppress the degradation of IkB (Fig. B-2), which retains N F - kB in the cytosol and therefore 175 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. prevents NF-kB translocating to the nucleus upon stimulation. More importantly, our data clearly demonstrated that IL-10 inhibits NF-kB DNA-Binding activity through inhibiting TNF-Induced translocation of IkB as shown by the immunostaining assay in figure B-4. Our data is in agreement with studies in human peripheral blood mononuclear cells (Wang et. al., 1995), human monocytic cell lines U937, THP-1 and human intestinal epithelial cell line HT-29 cells (Shottelius et al., 1999) that have demonstrated inhibition of NF-kB DNA-binding activity by IL-10 upon LPS and TNFa stimulation, respectively. However, our results disagree with conclusions from other studies that IL-10 can not block NF-kB DNA binding activities (Dokter et al., 1996 and Clarke et al., 1998). The discrepancies in these studies might arise from different cell lines and different LPS concentrations used. Taken together, our data strongly support one of the possible molecular mechanisms through which NF-kB activity is inhibited: by pretreatment with IL-10, the degradation of NF-kB inhibitor IkB upon stimulation with TNFa is down- regulated, thus, the subsequent translocation of released NF-kB in blocked. As a result, the DNA-binding activity of NF-kB is reduced. The inhibition of the degradation of IkB could be contributed by two mechanisms. One is due to the inhibition of IKK (IkB Kinase) activation, which phosphorylates IkB (Baldwin, 1996; DiDonato et al., 1996; Brown et al., 1995). Phosphorylated IkB are then rapidly polyubiquitinated and targeted to 26 S proteasome for degradation (Brown et al., 1995). Recently, it has been demonstrated that IKK activity is inhibited by IL-10 176 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. in U937 cells and to lesser extent, in THP-1 cells (Schottelius, et al, 1999). The other possible mechanism of inhibiting IkB degradation is through preventing ubiquitination of phosphorylated IkB, rather than inhibiting the phosphorylation of IkB by IKK. This potential mechanism remains to be explored in our future studies. To fully illustrate the molecular mechanism of IL-10 inhibition of N F - kB activation, it essential to study the apical molecular in this pathway, IL-10R1. Homodimeric IL-10 binds to IL-10 receptor complex composed of IL-10R1 and IL-10R2 (also known as CRF2-4)(Liu et al., 1993 and Ho et al., 1993). IL-10R1 is obligated high affinity ligand binding and in mediating signaling through Janus tyrosine kinase Jakl (Liu et al., 1993 and Ho et al., 1993). Whereas, IL-10R2 plays a critical role in IL-10 mediated signaling by recruiting Tyk2, that catalyzes trans phosphorylation of IL-10R1 chain and Jakl(Kotenko et al, 1997 and Spencer et al., 1998). Aggregation of IL-10 receptor complex upon IL-10 engagement induces activation of Jak-Stat signaling pathway, in which IL-10R1 associated Jakl and IL-10R2 associated Tyk2 are activated. Jakl and Tyk2 then activate transcription factors such as Statl, Stat3 and Stat5 (Finbloom and Winestock, 1995; Ho et al, 1995; Lai et al., 1996; Weber-Nordt et al., 1996 and Wehinger et al., 1996). It was of interest to study whether Jakl or Tyk2 are required in inhibiting N F - kB by IL-10 treatment. To analyze this, Jakl and Tyk2 negative cells lines U4 and U1 cell lines were transiently transfected with inducible IL-10R1 in the tet-on system and treated with TNFa, in the presence and obscene of IL-10. Our results indicated that Jakl or Tyk2 are not likely to be involved in inhibiting N F -k B translocation by IL-10 177 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. treatment since the blockages of p65 translocation upon IL-10 treatment were observed in both U4 (Fig. B-5) and U1 cells (Fig. B-6). However, these results do not preclude the possibility that Jakl and Tyk2 might have redundant function in inhibiting NF-kB activation. To clarify this possibility, cell line that has Jakl and Tyk2 double mutant should be used for the above studies. Nevertheless, our results are in conflict with the conclusion that Jakl is obligated in inhibit TNFa production in LPS-induced macrophages from Jakl knock-out mice (Meraz et al., 1996). This disparity might due to different stimulus, LPS and TNFa, used in these two studies. It has been shown that the extent of IL-10’s inhibitory effects depend on stimulus, since TNFa-induced NF-kB activity is reduced to a greater degree than that of LPS-induced in human monocytic cell lines (Schottelius et al., 1999). This implies that there could be differences in the paradigm of mediators in signal transduction pathways induced by these two stimuli. Therefore, it is possible that Jakl is indispensable in mediating LPS-stimulated pathways, whereas it not essential in inhibiting TNFa-induced NF-kB activation by IL-10 treatment. Previous studies have suggested that Stat3 participate in IL-10 effects on B cells and macrophages (Ho et al., 1995; Weber-Nordt et al., 1996; Stahl et al., 1995). However, there are conflicting results in terms of whether Stat3 is obligatory in for the anti-inflammatory functions of IL-10 (Ho et al., 1995, Weber-Nordt et al., 1996; Stahl et al., 1995; O’Farrell et al., 1998). Therefore, we intended to elucidate this inconsistency by mutagensis studies. By eliminating the two redundant Stat3 docking sites on IL-10R1 through mutating residues tyrosine-446 and tyrosine-496 178 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. to phenylalanines, our results indicated that Stat3 docking sites on IL-10R1 are not required IL-10-dependent inhibition of NF-kB activation upon TNFa stimulation (Fig. B-7). Our result is supported by the studies conducted by O’Farrell et al. (1998) where it was demonstrated that IL-10-dependent inhibition of TNFa production occurs in Stat3-independent manner. However, our results contradict with the report by Riley et al. (1999) where it is reported the opposite. These contradictory results might arise, again, as the result of different stimuli used in each study, such as LPS verses TNFa. In addition, different methodologies and cells lines, such as mouse primary macrophage cells (Riley et al., 1999), mouse J774 macrophage cell line (O’Farrell et al, 1998) and human cervical cancer cell line Hela cells (this report) are used. Furthermore, all these results can not rule out the possibility that Stat3 is involved in IL-10-dependent inhibition of NF-kB, considering the report that Statl can directly bind to TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) complex whereby suppressing NF-kB activation (Wang et al., 2000). To further delineate the critical functional domains in IL-10R1 involved in IL-10 dependent inhibition of NF-kB upon TNFa stimulation, the effect of deleting the last 28 amino acids at the carboxyl terminal of IL-10R1 was examined in this study. By introducing this deletion mutation of IL-10R1 to the cells, the inhibitory effect of IL-10 to NF-kB is abolished as demonstrated in figure B-8. Our result is supported by another study, in which mouse macrophage cells carrying murine IL-10R1 with the deletion of 30 amino acids in the carboxyl terminal failed to inhibit 179 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. IL-10 dependent inhibition of TNFa production by LPS-stimulation (Riley et al., 1999). Since the serine residues within the carboxyly terminus of the murine IL-10R1 are required for inhibition of TNFa production (Riley et al., 1999), the function of human IL-10R1 with all six serine residues mutated to alanines is currently undertaken in our lab. In summary, using IL-10R1 deficient Hela cells expressing wild type and various mutated human IL-10R1, the data presented herein clearly demonstrated that IL-10 inhibits NF-kB activation upon TNFa stimulation by inhibiting IkB degradation, and consequently, inhibiting NF-kB nuclear translocation and DNA-binding. The two receptor associated tyosine kinases Jakl or Tyk2 are not indispensible for the inhibition of NF-kB by IL-10 treatment. 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Creator Song, Yahui (author)

Core Title Cloning, characterization, anti -apoptotic molecular mechanism, transgenic and protein binding studies of mouse TOSO gene

School Graduate School

Degree Doctor of Philosophy

Degree Program Molecular Microbiology and Immunology

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Advisor Dubeau, Louis (committee member), Jacob, Chaim (committee member), Lai, Michael M.C. (committee member)

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