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Involvement of 3-phosphoinositide-dependent kinase 1 (PDK1) in the regulation of nitric oxide expression… Jian, Zhiqi 2005

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INVOLVEMENT OF 3-PHOSPHOrNOSITLDE-DEPENDENT KINASE 1 1PDK1) EN THE REGULATION OF NITRIC OXIDE EXPRESSION IN LPS-STIMULATED MACROPHAGES by ZHIQIJIAN Bachelor in Medicine, Sun Yat-Sen University of Medical Sciences, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA October 2005 © ZHIQI JIAN, 2005 ABSTRACT Macrophages are part of the innate immune system playing an important role in intestinal inflammation, and amplify the inflammatory response through the activation of Th-1 cytokines including nitric oxide (NO). Macrophages become activated as a result of exposure to microbial product lipopolysaccaride (LPS) as well as interferon-y from T cells. One of the aspects that attract researchers' interests most is the induction of inducible nitric oxide (iNOS) and increased NO production in these cells. PDK1 is a key enzyme in linking extracellular signals to multiple effector pathways. The mechanism by which LPS induces NO synthesis in murine macrophages is incompletely understood, and a role for PDK1 had not been previously investigated. In this study we demonstrate the involvement of PDK1 in the regulation of nitric oxide expression in LPS-stimulated Raw 264.7 macrophages. N-oc-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was used as an inhibitor of PDK1 signaling. The inhibition of PDK1 by TPCK led to the suppression of NF-KB activity, iNOS expression and NO production. What's more, Raw 264.7 macrophages transiently transfected with a kinase-dead PDK1 construct also showed decreased NF-kB reporter activity, iNOS protein expression and NO production. To further confirm our findings, we also created a stable cell line by transducing the lentiviral vectors expressing kinase-dead PDK1 into Raw 264.7 macrophages. The NF-KB pathway and the NO production were suppressed too in response to LPS compared to normal Raw 264.7 cells. Therefore, our results show for the first time that PDK1 is involved in NO expression in LPS-stimulated Raw264.7 macrophages and that this is associated with attenuation of LPS-mediated activation of the NF-KB pathway. TABLE OF CONTENTS ABSTRACT .' - 1 1 TABLE OF CONTENTS " i LIST OF FIGURES v i ABBEVIATIONS v i i ACKNOWLEDGEMENTS * CHAPTER 1 - INTRODUCTION 1 1.1 MACROPHAGES 1 1.1.1 Biology 1 1.1.2 Function 2 1.2 REACTIVE OXYGEN SPECIES 4 1.2.1 Free Radicals in Cell Biology .4 1.2.2 Nitric Oxide 4 1.3 NF-KB 7 1.3.1 Structure 7 1.3.2 Regulation 8 1.3.3 Function 9 1.4 SIGNALING 10 1.4.1 LPS Signaling 10 1.4.2 Inducible NO Synthase 12 1.4.3 PDK1 pathway 13 1.5 INFLAMMATORY BOWEL DISEASE 16 1.5.1 Pathology 16 1.5.2 Pathogenesis 16 1.5.3 The role of macrophages and nitric oxide 17 1.6 LENTTVIRAL VECTOR 18 1.6.1 LENTIVIRUS 18 1.6.2 LENTTVIRAL VECTOR STRUCTURE 19 iii CHAPTER 2 - MATERIALS AND METHODS .24 2.1 MATERIALS - - 2 4 2.1.1 Cell lines and cell culture 2 4 2.1.2 Reagents, Enzymes, and Chemicals 24 2.1.3 Plasmids - 2 4 2.2 METHODS 2 5 2.2.1 Cell Fractionation 2 5 2.2.2 Griess Assay 2 5 2.2.3 Immunoprecipitation 2 5 2.2.4 PDK1 Kinase Assay 2 6 2.2.5 Immunoblotting 2 6 2.2.6 Nuclear Preparation ; 2 ^ 2.2.7 Electrophoretic Mobility Shift Assay (EMSA) 27 2.2.8 Transient Transfecuon 2 8 2.2.9 Luciferase Assay 2 8 2.2.10 Lentiviral Vector Construction 2 8 2.2.11 Lentiviral Packaging 2 9 2.2.12 Zeocin Selection Raw 264.7 cells 29 2.2.13 Stable Cell line Selection 29 CHAPTER 3 - HYPOTHESIS 33 CHAPTER 4 - PDK1 inhibition by TPCK 34 4.1 RATIONALE 3 4 4.2 RESULTS 34 4.2.1 PDK1 activation by LPS stimulation in murine macrophages 34 4.2.2 TPCK inhibition of iNOS expression and NO production 34 4.2.3 TPCK inhibition of PDK1 activation 35 4.2.4 TPCK inhibition of NF-KB pathway 35 4.2.5 TPCK inhibition of NF-KB nuclear binding 36 CHAPTER 5 - PDK1 INHIBITION B Y TRANSIENT TRANSFECTION 42 5.1 RATIONALE 42 iv 5.2 RESULTS „ 5.2.1 iNOS induction and NO production in transfected Raw cells 5.2.2 PDK1 signaling in transfected Raw cells 5.2.3 NF-KB pathway in transfected Raw cells 42 CHAPTER 6 - PDK1 INHIBITION BY STABLE TRANSDUCTION 47 6.1 RATIONALE 47 6.2 RESULTS 47 6.2.1 NO production in transduced Raw cells 6.2.2 PDK1 signaling in transduced Raw cells 6.2.3 NF-KB pathway in transduced Raw cells CHAPTER 7 - CONCLUSION REFENRENCES LIST OF FIGURES Figure 1. LPS-induced iNOS expression pathway 21 Figure 2. PDK1 signaling 22 Figure 3. Schematic structure of HIV genome 23 Figure 4A. Lentiviral vector construction i 31 Figure 4B. Lentivral packaging 31 Figure 5. Zeocin selection of Raw 264.7 cells 32 Figure 6. Hypothesis 33 Figure 7. PDK1 activation by LPS stimulation 37 Figure 8. TPCK inhibition of iNOS expression and NO production 38 Figure 9. TPCK inhibition of PDK1 activation 39 Figure 10. TPCK inhibition of NF-KB pathway 40 Figure 11. TPCK inhibition of NF-KB nuclear binding 41 Figure 12. iNOS induction and NO production in transfected Raw cells 44 Figure 13. PDK1 signaling in transfected Raw cells 45 Figure 14. NF-KB pathway in transfected Raw cells 46 Figure 15. NO production in transduced Raw cells 49 Figure 16. PDK1 signaling in transduced Raw cells 50 Figure 17. NF-KB pathway in transduced Raw cells 51 vi ABBREVIATIONS Abbreviation Definition AP-1 activator protein-1 A P C antigen presenting cell BH4 tetrahydrobiopterin B L P s bacterial lipoproteins C a M calmodulin C D Crohn's Disease E G F epidermal growth factor E M S A electrophoretic mobility shift assay E V empty vector F B S fetal bovine serum F A D flavin-adenine dinucleotide F M N flavin mononucleotide FLTV human immunodeficiency virus K D kinase dead L P S lipopolysaccharide L T A lipoteichoic acid LTRs long term repeats L R R s leucine-rich repeats L V lentiviral vector IBD inflammatory bowel disease IFN-y interferon-y IGF-1 insulin-like growth factor-1 IKB inhibitor of KB I K K IKB kinase IL-1 interleukin-1 vii ILK integrin-linked kinase iNOS inducible nitric oxide synthase , IRAK interleukin-1 receptor associate kinase p70S6K 70-kilodalton ribosomal S6 kinase PDGF platelet-derived growth factor PDK1 3' phosphoinositide dependant kinase-1 PGNs peptidoglycans PI(3,4,5)P3 phosphoinositol(3,4,5)triphosphate PI(4,5)P2 phosphoinositol(4,5)biphosphate PI3-K phosphatidylinositol-3 kinase PKA protein kinase A PKB protein kinase B PKC protein kinase C MAF acrophage activating factor MAPK mitogen activated protein kinase N F K B nuclear factor KB NLS nuclear localization sequence NMA N^monomethyl-L-arginine NO nitric oxide RRE Rev Response element SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis TGF-p transforming growth factor-fj TH1 helper T cell, type 1 TH2 helper T cell, type 2 TTF Toll/TL-IR TLR4 Toll like receptor 4 TNF-a tumor necrosis factor a TPCK N-a-tosyl-L-phenylalanyl chloromethyl ketone TRAF-6 tumor necrosis factor receptor associate factor-6 UC ulcerative colitis viii VSV-G vesicular stomatits virus protein G WT wild type ACKNOWLEDGEMENT I thank Dr. Alice Mui and Rupinder Dhesi from her lab for the help in constructing the lentiviral vector. CHAPTER 1 - INTRODUCTION 1.1 MACROPHAGES 1.1.1 Biology Macrophages are a population of ubiquitously distributed mononuclear phagocytes. They go through four distinct cell stages from pluripotential stem cells to reach maturity, designated monoblasts, promonocytes, monocytes and macrophages, which are defined by their specific cell surface markers and endogenous peroxidatic activity[l]. Mature monocytes, which represent about 5% of white blood cells, are released from bone marrow and are maintained in the circulation for up to 3 days[2]. Their wide tissue distribution makes these cells well suited to provide an immediate defense against foreign elements prior to leukocyte immigration[l]. Macrophages from different tissues exhibit a wide range of phenotypes with regard to both their morphology and function; the local environment has a profound effect on macrophage differentiation[3]. This heterogeneity contributes to the wide array of functions of macrophages including homeostatic, immunological, and inflammatory functions. Macrophages can be activated by two major ways: (i) as a consequence of their interaction with microorganisms or their products such as endotoxins and (ii) via soluble mediators released by antigen termed as macrophage activating factor (MAF). There are two stages of macrophage activation, the first being a "primed" state in which macrophages exhibit enhanced MHC class II expression, antigen presentation, and oxygen consumption, but reduced proliferative capacity. Primed macrophages respond to secondary stimuli to become "fully activated," a state defined by their inability to proliferate, high oxygen consumption, killing of facultative and intracellular parasites, tumor cell lysis, and maximal secretion of mediators of inflammation, including TNF-a, PGE2, IL-1, IL-6, reactive oxygen products, and nitric oxide.[4] 1.1.2 Function Macrophages play a major role in maintaining host integrity by their involvement in homeostasis, host defense and immunity[5]. They participate in both specific immunity via antigen presentation and interleukin-1 production[6] and nonspecific immunity against bacterial[7], viral[8], and fungal pathogens [9], as well as neoplasm [10]. They detect infectious agents by the pattern-recognition molecules expressed on cell surface. These molecules serve as both opsonins (mannose-binding protein) and cell surface-bound receptors (mannose receptor) to recognize a broad range of pathogens through terminal sugars.[3] Phagocytosis One of the defining features of macrophages is their phagocytic capacity[3]. Phagocytosis constitutes the first line of immune defence against pathogenic bacteria that have penetrated the epithelial barrier[5]. The uptake of large particles begins with the binding of receptor on the phagocyte membrane with specific molecules, ligands localized on the surface of the particle to be engulfed. At the next stage, the ligand-receptor complex induces rearrangements in the actin cytoskeleton leading to pseudopodial formation and internalization of the particle. [11] A number of phagocytosis-promoting receptors have been identified on macrophages, including mannose receptor, CD14, which recognizes surface components of bacteria, including lipopolysaccharide (LPS), scavenger receptor A, and CD36, CD68, which are involved in phagocytosis of apoptotic cells[12]. Unlike the uptake of infectious agents that cause pro-inflammatory responses, macrophages that have ingested apoptotic cells actively suppress the release of inflammatory cytokines triggered by LPS[3]. Secretion More than 100 different well defined molecules are secreted by macrophages in 2 response to pathogens and "danger" signals, which fall into a number of broad categories: cytokines, chemokines, growth factors, eicosanoids, enzymes and enzyme inhibitors, clotting factors and complement components, plasma binding proteins etc [13]. They also release reactive metabolites of oxygen and nitrogen and proteases that degrade the extracellular matrix.[14] The secretion of these factors depends on the type of stimulus, macrophage type and location. Cytokines are a group of highly conserved hormone-like polypeptides produced by different cells which act in an autocrine, paracrine or endocrine fashion to modulate inflammatory and immunological reaction[15]. They are extremely potent often being active at nanomolar or picomolar concentration 16]. The list of cytokines is long and growing, among which interleukin (IL)-l, IL-6, EL-12, JLL-18, TNF-alpha are studied extensively. The role that cytokines play in the regulation and modulation of immunologic and inflammatory processes is striking. They act cooperactively to control normal physiologic activity and disease-related threats to homeostasis[17]. They can drive the initial inflammatory response; encompass leukocyte mobilization, coagulation alterations, and tissue repair in response to an invading organism[18]. Yet an overexuberant proinflammatory cytokine response results in overreaction to the primary insult, which in turn does harm to the body. High levels of cytokines have been associated with a myriad of pathophysiologic processes, such as sepsis[19], asthma[20], inflammatory bowel disease[21, 22], to name a few. Wound Healing Macrophages are required for angiogenesis and wound healing[23]. The response to injury can be classified into three stages: inflammation, proliferation and regeneration. Macrophages play a major role not only in the initial phases of the inflammatory response but also in the growth phase, by secreting angiogenic and fibrogenic growth factors that repair damaged tissue such as transforming growth factor-P (TGF-P), epidermal growth factors (EGFs), platelet-derived growth factors (PDGFs) and insulin-like growth factor-1 (IGF-1)[5, 16]. The cytokines and chemokines at the site 3 of inflammatory responses secreted by macrophages influence the expression and function of a number of cell adhesion molecules on the surface of endothelial cells, which may selectively recruit cell types that are needed for wound healing process and which also express specific ligands[5]. In addition, macrophages secrete several complement inhibitors and a wide range of proteases and proteinase inhibitors that facilitate reduction of inflammation, tissue remodelling[16, 24]. 1.2 Reactive Oxygen Species 1.2.1 Free radicals in Cell Biology In aerobic cells, free radicals are constantly produced mostly as reactive oxygen species, including nitric oxide and related species, which commonly exert a series of useful physiological effects. However, there is reasonable evidence to suggest that free-radical-mediated tissue damage is a significant event in the pathogenesis of many diseases including atherosclerosis, autoimmune diseases, inflammation, reoxygenation injury, and malignant diseases. [25] 1.2.2 Nitric Oxide Chemistry Nitric oxide NO with molecular weight 30 is the smallest ubiquitous diffusible cell signaling molecule.[25] Species that will react with NO are oxygen, transition metals (such as Fe 2 + haem groups in the enzyme guanylate cyclase) and other radicals (e.g., superoxide). Reactive nitrogen species derived from NO, such as nitrosonium (NO+), nitroxyl (NO"), nitrogen dioxide ( N O 2 ) , nitronium (N0 2 +) and peroxynitrite (ONOO"), are the molecules most likely to be responsible for the deleterious effects attributed to NO.[26] NO has multiple important physiological roles, such as vasodilation and neurotransmission. The chemistry of these homeostatic functions is due to NO 4 binding certain transition metal ions. For example, NO binds the active site of cyclic GMP, which results in its activation, leading to further production of cGMP. This lowers intracellular free Ca 2 + which, in turn, relaxes smooth muscle. The blood vessel dilates and blood pressure is lowered.[27] Cell and Tissue Effects of Nitric Oxide NO is an important intracellular and intercellular signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems[28]. It acts as a biological mediator similar to neurotransmitters in the neuronal system; regulates blood vessel tone in vascular systems, and is an important host defence effector in the immune system. On the other hand, overproduction of NO has been associated with many pathological processes, particularly inflammatory disorders due to redundant free radicals acting as cytotoxic agents[29, 30]. Nevertheless, there are conflicting reports of the effects of NO, being either proinflammatory or anti-inflammatory. The consensus is that it should be taken into consideration of timing, location, and rate of production of NO and the relevant targets affected[31]. Nitric Oxide in Macrophages Human monocytes and macrophages can express functional NOS2 at the protein level. When NOS was expressed and inhibited pharmacologically in vitro, macrophages' antimicrobial activity decreased[32]. A lot of work has been done to test the proposition that NO production is one of the major antimicrobial mechanisms of macrophages, (i) In cell lines and inbred mouse strains, NOS2 expression is correlated with host resistance to microbial growth[33-36]. (ii) Although in some settings the inhibitors benefited the host, the overwhelming majority of studies show that inhibition of high-output NO pathway in macrophages worsens the course of diseases caused by an impressive array of phyla-viruses, bacteria, fungi, protozoa, and 5 helminths[33, 37-40]. (iii) Experiments using exogenous NO to treat infected cells[33] or infectious agents in host cell-free systems[41, 42] all support the notion that NO can be both necessary and sufficient for the antimicrobial actions of NOS2. What's more, host resistance decreases in genetically engineered mice deficient in NOS2 (NOS2"/_) although activation and the respiratory burst of macrophages, lymphocyte ontogeny, and leukocyte migration are unimpaired [43]. NO's diffusional and frans-acting properties favour the extracellular killing against tumor cells. NG-monomethyl-L-arginine (NMA) blocked the cytotoxic action of IFNy/LPS-activated primary mouse macrophages against guinea pig L10 hepatoma or mouse L1210 lymphoma cell lines mimicked by NO gas or acidified N O 2 " , which is absent in macrophages from NOS2"'" mice[44]. However, not all tumor cells inhibited by activated macrophages succumb via NO, and the sensitivity or resistance of tumor cells to macrophages varies as well. This diversity of cytotoxic mechanisms and variability in tumor cell susceptibility have yet to be clarified. Nitric Oxide Synthesis The enzyme that drives the oxidation of L-arginine to yield NO and the by-product L-citrulline is NO synthase (NOS), a flavin-containing hemoprotein[45] encoded by three separate genes located at 7, 12, 17 human chromosomes [25]. At least three different NOS isoforms are identified, and they fall into two distinct types, constitutive and inducible[46] . The constitutive form is regulated at the level of enzyme activity by calcium and calmodulin binding and is present in the nervous system as neuronal NOS (nNOS or NOS-1) and in the vascular endothelium as endothelial NOS (eNOS or NOS-3).47] Inducible nitric oxide synthase (iNOS or NOS-2) is produced in response to microbes, cytokines, and other activating stimuli in many cell types including macrophages[48, 49] and epithelial cells[50, 51]. All three mammalian isoforms are thought to catalyze NO by the same biochemical pathway[52] . One molecule of L-arginine is oxidized to yield one molecule each of NO and and L-citrulline, in which 1.5 molecules of NADPH and two molecules of dioxygen are consumed. Mammalian NOSs only make NO when homodimeric. Each subunit is thought to attach at least five other molecules besides the co-substrates and the partner monomer, i.e. heme, tetrahydrobiopterin (BH4), calmodulin (CaM), and the flavin mononucleotide (FMN) and flavin-adanine dinucleotide (FAD)[27, 52]. NOS1 and NOS3 produce NO for minutes per response episode; in contrast, NOS2 in mouse macrophages can produce NO for as long as 5 days. This explains the ability of NOS2 to release orders-of-magnitude more product than the other isoforms[52]. The induction of iNOS is regulated mainly at the transcriptional level by transcription factors, but also at posttranscriptional, translational and posttranslational levels through effects on protein stability, dimerization, phosphorylation, cofactor binding and availability of oxygen and L-arginine as substrates [28]. 1.3 NF-KB 1.3.1 Structure One of the most ubiquitous eukaryotic transcription factors that regulate expression of genes involved in controlling cellular proliferation/growth, inflammatory responses, cell adhesion, etc. is nuclear factor-kappa B (NF-KB)[53, 54]. The functionally active NF-KB exists mainly as a heterodimer consisting of subunits of Rel family, and is regulated via shuttling from the cytoplasm to the nucleus in response to cell stimulation[55]. Mammals express five Rel proteins that belong to two classes. The first class includes RelA, c-Rel and RelB, proteins that are synthesized as mature products and do not require proteolytic processing. The second group is encoded by the Nfkbl and Nflcb2 genes, whose products are first synthesized as large precursors, pi05 and pi00, respectively, that require proteolytic processing to produce the mature p50 and p52 NF-KB.[56] NF-KB containing RelA or c-Rel are normally sequestered in an inactive cytoplasmic complex by binding to an inhibitory protein, IKB.[57] p65/p50 heterodimers were the first form of NF-KB to be identified and are the most abundant 7 in most cell types[58]. The Rel proteins differ in their abilities to activate transcription, such that only p65/RelA and c-Rel were found to contain potent transcriptional-activation domains among the mammalian family members. The IKB family includes hcBa, IKB(3, IKBy , IKBE, Bcl-3, the precursors of NF-KB 1 (pl05), and NF-KB2 (plOO), and the Drosophila protein Cactus[55, 58, 59]. IKB a was the first IKB family member to be cloned and is still the best characterized. Only IKBCX, LcBp, IKBE, contain N-terminal regulatory regions, which are required for stimulus-induced degradation, the key step in NF-KB activation[58]. IKBS also play an important role in termination of NF-KB activation. Newly synthesized IKB a enters the nucleus and binds to N F - K B , thus facilitating the dissociation of NF-KB from DNA and causing the re-exportation to cytoplasm[60]. 1.3.2 Regulation NF-KB is activated by exposure of cells to a wide variety of different stimuli, including pro-inflammatory cytokines such as TNF-a and LL-1, T- and B-cell mitogens, bacteria, and bacterial lipopolysaccharide, viruses, viral proteins, double-stranded RNA, and physical and chemical stresses[55, 61]. The canonical pathway of NF-kB activation is well established. Upon cells receiving potent activating stimuli, IKBS (especially IKBOI) is quickly phosphorylated by a large kinase complex, termed iKB-kinase (IKK), which intergrates signals from multiple pathways. Such inducible IKB phosphorylation occurs at serine 32 and 36 in IKBCI. Phosphorylation leads to the immediate recognition of IKBOI by the F-boxAVD40 E3RSI,cB/p-TrCP, which consequently results in the polyubiquitinylation of IicBa primarily at lysine 21 and 22 by an SKpl-Cullin-F-box (SCF)-type E3 ubiquitin ligase[58]. This modification then targets IKBCI for rapid 26S proteasomal mediated degradation. The nuclear localization sequence (NLS) of NF-KB is then exposed leading to the binding with karyopherins and translocation of NF-KB to the . 8 nucleus[58], where it induces the transcription of a large number of target genes that normally encode cytokines, cell adhesion molecules, and growth factors through binding the ris-acting KB element Before p65 becomes transcriptionally active it requires post-translational modifications including both phosphorylation and acetylation. These are crucial for p65 mediated transactivation. CBP/p300 provides acetylation on p65 on lysine 218, 221, and 310, with lysine 310 being an essential step for full transcriptional activity of p65[62, 63]. Similarly, phosphorylation at serine 276, 311, 529, 535, 536 have all been shown to be required for full transcriptional activity of p65. Several protein kinases have been implicated in these processes including protein kinase A (PKA) for Ser276[64, 65], protein kinase C zeta (PKCQ for Ser311[66], protein kinase CK2 for Ser529[67], calmodulin-dependent kinase IV for Ser535[68], and IKKp, protein kinase B (PKB) for Ser536[69, 70]. Besides the classical pathway, there are at least two additional activation pathways reported. One is believed to require phosphorylation of IKBCX at Tyr 42[71, 72]. Although the exact protein tyrosine kinase involved is not known, certain members of the Src family were proposed to be responsible for this phosphorylation^]. The second atypical activation pathway occurs in IKBCX degradation induced by UV radiation (254nm) via the 26S proteasome[58]. This process is not mediated by phosphorylation of Ser 32 and Ser 36 or Tyr 42. The exact mechanism is unknown. In both these alternative pathways, NF-kB activation is considerably slower and weaker than the classical pathway. 1.3.3 F u n c t i o n NF-KB regulates the transcription of an array of genes, some of which promote inflammation, leukocyte migration and activation, whereas others act as potent inhibitors of apoptosis, thus representing a central regulator of innate and adaptive 9 immune responses. Besides, NF-KB is also important in control of cell proliferation, oncogenesis and cell transformation[73]. Thus, inappropriate regulation of NF-KB is directly involved in a wide range of human disorders, including a variety of cancers[74], inflammatory bowel disease[75], and numerous other inflammatory conditions. In this thesis, we will focus on the discussion of the immune responses, especially in the gut. NF-KB is a key regulator of the inducible expression of many genes associated with immune function in the gut. For instance, in lymphocytes, epithelial cells and monocytes, NF-KB can induce cytokines such as IL-1, IFN-y, IL-2, IL-6, IL-8, IL-12p40[75]. On the other hand, NF-kB itself is extensively up- and downregulated by a wide variety of exogenous stimuli that modulate immune function, thus providing a positive or negative feedback mechanism. For example, NF-KB transactivates the iNOS promoter in response to LPS giving rise to increased production of NO, which in turn has been reported to inhibit NF-KB activation in endothelial cells[76]. The targeted disruption of various genes encoding NF-KB subunits has provided much insight into the function of specific subunit of NF-kB in the immune system. The phenotype of p65 (RelA) knockout mice was dramatic. These animals died during embryonic development. A loss of inducibility of NF-kB regulated genes (GM-CSF, IKBCO is also revealed and the cultured T cells from these mice showed strikingly reduced proliferative responses. However, mice lacking the p50 subunit developed normally but had severe defects in immune cell function[77]. B cells of these mice had an impaired ability to produce antibodies and to proliferate upon LPS challenge[75]. RelB"7" mice showed a complex pathological phenotype, which is the result of multiple defects in the adult immune system from day 8-10[78]. Mature T and B lymphocytes of mice lacking c-Rel had an impaired responsiveness to mitogenic stimuli like anti-CD3 or anti-IgM, respectively[75]. 10 1.4 Signaling 1.4.1 LPS Signaling The bacterial endotoxin LPS is a predominant, integral structural component of the outer membrane of Gram-negative bacteria and one of the most potent microbial initiators of inflammation[79]. Macrophage activation requires recognition of LPS via LPS binding protein. Binding of microbial products to CD 14 triggers production of different cytokines such TNF-a, IL-1, and IL-6, which in turn mediate inflammation through interactions with various target cells. Membrane-bound CD14, a glycosyl-phosphatidyl inositol-anchored protein, has been implicated in cell activation processes involving other products of microbial pathogens, including lipoarabinomammans, peptidoglycans, and outer membrane lipoproteins[80]. Because of the fact that CD14 is devoid of an intracellular domain, LPS responsiveness was not solely dependent upon CD 14. In CD14-deficient mice, LPS can activate monocytes to produce TNF-alpha and IL-6, albeit at concentrations two to three times higher than those required for wild-type mice[81]. Thus, LPS and other microbial products most likely employ different CD14-associated receptors to transduce their signals. The transmembrane protein that acts with CD 14 to generate a transmembrane signal linked to LPS-induced cell activation turned out to be Toll-like receptor 4 (TLR4), an ortholog of the TLRs that mediate innate immunity in Drosophila{%2]. TLRs represent a growing family of transmembrane proteins characterized by multiple copies of leucine-rich repeats (LRRs) in the extracellular domain and a cytoplasmic Toll/IL-IR (TIR) motif[83]. TLRs are evolutionarily conserved and their congeners have been found in insects, plants, and mammals. Consistent with their role in pathogen recognition and host defense, mammalian TLRs are strategically expressed in monocytes/macrophages, neutrophils, dendritic cells, intestinal epithelial cells and endothelia cells - cell types that are immediately accessible to microorganisms upon n infection. TLR2 and TLR4 have also been found in B and T cells, possibly indicating their role in modulating adaptive immune responses[83, 84]. In humans, nine full length TLR sequences have also been deposited in GenBank, while six other members remain partially characterized[83, 85, 86]. Among the nine known mammalian TLRs, TLR2 and TLR4 have been extensively characterized. TLR4 is the primary signal transducer for LPS and lipoteichoic acid (LTA); while TLR2 responds to peptidoglycans (PGNs), cell wall components of Gram-positive bacteria[87], and low level of contaminating bacterial lipoproteins (BLPs) in commercial LPS preparation[83, 88]. The exact ligand binding mechanism by which LPS interacts with TLR4 is not clear. The discovery of MD-2, a nonmembrane-spanning molecule that physically associates with TLR4 and is required for optimal LPS-induced, TLR4-mediated signaling[89], just provided another potential candidate[79]. Upon engagement with LPS, TLR4 is activated and undergoes dimerization, resulting in a conformational change in the cytoplasmic TLR domain and subsequent recruitment of an adapter named MyD88[85, 86]. MyD88 associates with the TLR via a homophilic interaction using the TLR domains. The death domain of MyD88 then recruits a downstream serine-threonine kinase known as IL-1 receptor-associated kinase (IRAK) to the receptor complex[86]. IRAK is then autophosphorylated and dissociated from the receptor complex and recruits TNF receptor-associated factor 6 (TRAF6) that in turn activates downstream kinases. Subsequently, IKB is phosphorylated and degraded, leading to NF-kB transactivation. However, the mechanism by which IKK complex is activated is not well understood. In addition to NF-KB activation, TLRs can also initiate mitogen-activated protein kinase (MAPK) signaling cascades and thus activate multiple transcription factors, including activator protein-1 (AP-1) and Elk-1 [83, 85, 90]. 1.4.2 iNOS Signaling The induction of iNOS is mainly regulated at the transcriptional level. The promoter 12 region of the mouse iNOS gene contains several transcription factors binding sites such as NF-KB as well as Jun/Fos heterodimers, some C/EBT, CREB and the STAT family of transcription factors, within its proximal and distal regions[91-93]. The human iNOS gene promoter contains sequences homologous to mouse proximal and distal regions[94]. The degradation of IKB, translocation of free NF-KB to the nucleus is an important mechanism in regulating NO production by iNOS [36], iNOS can also be induced after exposure of cells to IFN-y by the Jak-STAT signaling pathway. Meanwhile, the production of negative regulator suppressor of cytokine signaling-1 (SOCS-1) inhibits cytokine signals that activate this pathway[94]. Interestingly, NO has biphasic effects on its own synthesis by modulating iNOS mRNA expression[95]. The negative feedback regulation in macrophages and hepatocytes is mediated by the inhibition of NF-KB activation. 1.4.3 PDK1 Pathway 3-Phosphoinositide-dependent kinase 1 (PDK1) belongs to the AGC superfamily of serine/threonine kinases. PDK1 is a 556 amino-acid protein with an N-terminal catalytic domain and a C-terminal Pleckstrin Homology (PH) domain, first identified as the upstream activation loop kinase of protein kinase B (PKB, also known as Akt) [96, 97]. The PH domain binds with high affinity to the PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2 (P IP2 ) second membrane bound messengers that are generated by phosphatidylinositol 3-kinase (PI3-K) following stimulation of cells with growth factors[97, 98]. PDK1 phosphorylates PKB at threonine 308, a critical phosphorylation site in the activation loop for PKB activation. After this discovery, a lot of work has been done on the PDK1 signaling pathway, leading to the identification of more PDK1 downstream targets, including the 70-kilodalton ribosomal S6 kinase (p70 S6-K), PKC isoenzymes, and perhaps PKA, which have been previously shown to be activated downstream of PI3-K. p70 S6K is an enzyme thought to initiate protein synthesis. Phosphorylation of p70 by PDK1 is probably regulated in vivo by the accessibility of the p70 activation loop, which is controlled by 13 multiple, proline-directed phsphorylations within an autoinhibitory pseudosubstrate domain in the carboxy-terminal tail and by phosphorylation of threonine 412, the equivalent of PKB's threonine 473[99-101]. PDK1 and PKC^ associate in vivo via their catalytic domains, and coexpression with PDK1 enhances PKCt, activity and activation-loop phosphorylation in a manner sensitive to the PI3-K inhibitor wortmannin[102, 103]. In vitro, PDK1 catalyzes direct phosphorylation of the PKCt; activation loop, accompanied by a six-fold increase in PKC^'s kinase activity[101]. The conventional PKC isoforms-a, pi, pil, and y-are contain the highly conserved carboxy-terminus, which decides the catalytic competence of the kinase. Phosphorylation of these sites is catalyzed by intramolecular autophosphorylation, which can happen only after activation loop phosphorylation by another kinase[101]. PDK1 can phosphorylate the activation loop site of PKCpn in vitro. Although this phosphorylation does not alter its activity, it provides a conformational change that confers carboxy-terminal autophosphorylation. Coexpression of PKCpn with a kinase-inactive mutant form PDK1 leads to the accumulation of unphosphorylated and inactive PKCpII[104]. Thus, PDK1 is likely to be the kinase response for the constitutive, early phosphorylation of the activation loop in PKCpII, and probably also in the other conventional PKCs[101]. The phosphorylation of conventional PKCs is independent of PI3-K, suggesting that 3'-phosphoinositides may be entirely dispensable for PDK1 signaling in some cases[105]. Similarly, the phosphorylation of PKB/Akt by PDK1 is also regulated by the conformation of Akt. The engagement of the PH domain on the membrane by binding P I P 3 / P I P 2 relieves autoinhibition of the active site, allowing PDK1 to access Thr308 on the activation loop[106]. PDK1 function is also regulated by cellular relocalization. Previous studies have indicated that stimulated cells showed a PH domain-dependent relocalization of PDK1 from the cytosol to the plasma membrane[107, 108]. PI3-Kinase PI3-K refers to a family of proteins that are capable of phosphorylating the D-3 14 position of the inositol ring of phosphoinositide lipids. PI3-Ks can be divided into three main classes on the basis of their structure and specificity for substrate. Class I PI3-Ks phosphorylate PI, PI(4)P and PI(4,5)P2, generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3, the latter two are generally absent from resting cells. The prototypical class I A PI3-K is a heterodimer consisting of an 85 kDa regulatory subunit and a catalytic 110 kDa subunit. The p85 subunit facilitates the protein-protein interactions wither via protein tyrosine phosphate-binding SH2 domains or SH3 domains and/or proline rich regions [27]. PI3-K has been shown to be a pivotal lipid kinase involved in a diverse array of cellular responses including cell survival, mitogenesis, membrane trafficking, glucose transport, superoxide production as well as chemotaxis. The role of PI3-K/Akt signalling pathways in carcinogenesis process is well established. Recently, there is strong evidence for the involvement of this kinase in regulating immune response. PI3-K is activated in response to LPS, either by direct interaction with MyD88[109] or downstream of IRAK[110]. In either case, activation of this pathway appears to be essential for cytokine and NO production; given that the PI3K inhibitors wortmannin and LY294002 completely blocked their production from LPS-induced bone marrow-derived macrophages (BMmcps) and BMMCs[ll l] , However, experiments using other cell models yield strikingly different results. Park et al. have shown that PI3-K inhibitors enhanced LPS-induced iNOS expression and activity in murine macrophages[112]. Thus, one should keep in mind that the interaction between iNOS and PI3-K may vary depending on the cell model used[27]. PKB/Akt PKB/Akt is a serine/threonine kinase that is itself activated by two distinct phosphorylation events, Thr308 in the activation loop and Ser473 near the carboxyl terminus[113]. PKB has a carboxy-terminal catalytic domain and an amino-terminal, non-catalytic segment that contains PH domain. As discussed earlier, PDK1 is the 15 widely accepted upstream kinase for Thr308; however, the kinase responsible for Ser473 phosphorylation is less clear. Recently, the intergrin-linked kinase (ILK) has been shown to increase Ser473 phosphorylation[114-117]. PKB has been demonstrated to phosphorylate a number of proteins, particularly those involved in regulating glucose metabolism and cell survival[27, 118]. Besides, PKB phosphorylates IKK-a, ultimately leading to the activation of NF-KB[119]. The role of PKB in the immune response is largely unexplored, mostly linked to the activation of N F - K B . 1.5 Inflammatory Bowel Disease 1.5.1 Pathology Inflammatory bowel disease (D3D) is one of the most common chronic gastrointestinal illnesses in North America. There are about 10,000 new cases of IBD each year in Canada. D3D is a collective term referring to a complicated condition, including Crohn's disease (CD), ulcerative colitis (UC), microscopic colitis, and indeterminate colitis, that affects the intestine and several extraintestinal sites. CD and UC are the two major types, which share several characteristics including edema, loss of epithelial barrier, and gut leukocyte infiltration[120]. The current mainstream treatment for IBD is 5-ASA compounds which target activation of the transcription factor N F - K B , and modulate lipid inflammatory mediators. 1.5.2 Pathogenesis The pathogenesis of IBD is multifactorial. Affected individuals often have a genetic predisposition to develop CD or UC. After disruption of the gastrointestinal mucosal barrier, a luminal antigen causes ongoing activation of the mucosal immune system, which leads to tissue damage and the clinical features of IBD.[121] 16 Monozygotic twins' studies have shown a significantly higher concordance rate than dizygotic twins, indicating a genetic predisposition to L3D. Several genes on different chromosomes have been linked to the development of CD and UC[122, 123]. A second concept is the requirement of luminal flora for IBD to develop in a susceptible host[124]. However, there has not been one organism, or one group of organisms that has been found responsible for experimental colitis. In addition, a group of bacteria termed probiotic baciteria have been found actually to be protective[125]. In a genetically predisposed host, environmental triggers can cause a persistent and excessive activation of the mucosal immune system by altering the luminal flora or disrupting the mucosal barrier. In CD, the mucosa is dominated by typical type 1 helper-T cells, which play an important role in cellular immunity. The following production of cytokines such as TNF-a, IFN-y, IL-2 by activated macrophages results in inflammation and consequent tissue destruction. In contrast, the mucosa in UC is characterized by an atypical type 2 helper-T cells which produce IL-5, TGF-p, IL-13 leading to crypt abscesses, eventually tissue destruction. 1.5.3 The role of macrophages and nitric oxide Macrophages are perceived to be important players in the pathogenesis of IBD. There is a large population of them in normal intestinal mucosa where they represent the major APC capable of determining the type of T cell responses. They can also amplify the inflammatory response through the elaboration of IL-6, IL-8, IL-12, TNF-a as well as nitric oxide. The role of NO in IBD is controversial. iNOS expression and activity, and subsequently NO production, are generally increased in IBD. One hypothesis for the mechanism of NO-induced cellular damage is that NO interacts with oxygen radicals such as superoxide to produce peroxynitrite. This then reacts with tyrosine to form nitrotyrosine in cellular proteins.[30] The correlation of NO production with UC disease activity is well established[126-128], however, while some of studies support 17 such a correlation in CD[129-131], others do not[132, 133]. Studies using genetically modified animals were also controversial. While the majority show improvement of experimental IBD with iNOS inhibition[134-139], there are also a significant number of reports of exacerbation of disease with inhibitors[140-142]. Similarly, studies using iNOS-deficient mice in colitis model have shown improvement 143, 144], worsening[145, 146], or no effect on disease at all[145, 147]. The contradiction arising from these studies may due to the difference in species, strains, housing conditions, models and execution[148, 149]. Despite the discrepancies, a more or less definite assumption seems to be reached, that is that selective inhibition of iNOS may reduce, to some extent, the tissue damage observed following chronic up-regulation of this isoform in the settings of chronic colitis. On the contrary, the early inhibition of NO production during acute colitis produces equivocal results, indicating a protective role of either constitutive or inducible isoforms in the settings of an acute mucosal insult[150]. 1.6 Lentiviral Vector System 1.6.1 Lentivirus Gene transfer is a common technique in medical research, which may result in genetically transformed cells and individual organisms. Retroviral vectors have been employed for delivery of genetic material into cells for over twenty years, with the first reports dating back to the early 1980s. Vectors based upon human immunodeficiency vims (HIV) are noted for their ability to infect non-diving cells. The first lentiviral vector that had a quantitative titer was developed in 1990[151]. The lentiviruses have gained more and more ground as gene delivery tools, particularly in the past eight years. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis vims (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious[152]. Lentiviruses are a genus of the family RETROVIRIDAE consisting of non-oncogenic retroviruses that produce multiorgan diseases characterizing by long incubation periods and persistent infection. Retroviruses are RNA viruses. The virus particle contains two identical copies of genomic RNA along with viral and cellular proteins[153]. Besides the conserved structural and enzymatic genes encoding for the polyproteins Gag, Pol, and Env shared in retrovirus family, HIV's genome encodes for two regulatory genes, tat and rev, and four accessory genes, vif, vpu, vpr and nef, all thought to be intimately involved in viral pathogenesis[153]. The viral RNA is flanked on either side by elements called long terminal repeats (LTRs). The LTRs harbor the c«-acting promoter, upstream enhancers as well as polyadenylation signals essential for viral transcription. Additional m-acting elements include signals (acceptor and donor sites) for proper splicing of mRNAs, the packaging sequence (V) that allows the viral RNA to be specifically packaged into the virus particle and primer-binding sites for the initiation of reverse transcription in the recipient cell[153]. (see Figure 3) 1.6.2 Lentiviral vector structure The design of viral vectors is based on the separation of cw-acting sequences required for the transfer of the viral genome to target cells from the trans-acting sequences encoding the viral protein[154, 155]. Vector particles are assembled by viral proteins expressed in trans from construct(s) devoid of most viral cw-acting sequences (packaging constructs). The viral ds-acting sequences are linked to an expression cassette for the transgene (transfer vector construct). Both types of constructs are introduced in the same cell to produce vector particles. As the particles can only encapsidate and transfer the vector construct, the infection process is limited to a single round (transduction). The current lentiviral vector is a three-plasmid-based system that includes a helper construct, an envelope-encoding construct, and the transducing vector[156]. In this system, the helper construct lacks the viral 3' and 5' LTRs, V sequences, envelope-coding regions, and expresses the essential viral protein Gag, Pol. The envelope is from a different virus, most often the VSV for 19 pseudotyping, allows entry into a wide spectrum of target cells[156-159]. The transducing vector or transfer vector contains cw-acting sequences of HTV-1 necessary for reverse transcription and integration (3' and 5' LTRs and primer binding site), packaging sequences, splice signals, Rev response element (RRE) for nuclear export and unique restriction sites for cloning the transgene of interest. In addition to these m-acting sequences, an internal CMV promoter has been incorporated to facilitate the transcription of transgene in the absence of viral accessory proteins in the recipient cell. 20 Figure 1. LPS-induced iNOS expression pathway. 21 Figure 2. PDK1 signaling 22 5'LTR Gag VDT vif VDU nef 3'LTR \ Pol Env r j - « - a Figure 3. Schematic structure of OTV genome 23 C H A P T E R 2 - MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 Cell lines and Cell Culture Raw 264.7 cells were acquired from the American Type Cell Culture (ATCC, Manassas, VA). Raw 264.7 were maintained in DMEM (HyClone, Logan, Utah) supplemented with 3.7g/L sodium bicarbonate, 50U/ml penicillin, 50ug/ml streptomycin (Life Technologies), and 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, Utah). Al l cell lines were grown at 37°C with 5% C0 2 . 2.1.2 Reagents, Enzymes, and Chemicals N-oc-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was purchased from Calbiochem (San Diego, California); Lipopolysaccharide (LPS, from Escherichia coli.) was a kind gift of Dr. Alice Mui (University of British Columbia, Vancouver, B.C.). TPCK stock solution was made up in dimethylsulfoxide (DMSO) and LPS in sterile water. Al l restriction enzymes were purchased from New England Biolabs (Mississauga, Ontario, Canada). Superfect transfection reagent was purchased from Qiagen (Mississauga, Ontario). 2.1.3 Plasmids The empty vector (pCMV5-Myc) and PDK1 kinase-dead (KD) plasmid (pCMV5-Myc-PDKl) were kind gifts from Dr. Jongsun Park (Friedrich Miescher Institue, Basel, Switzerland). The KD vector contains a point mutation (K61Q) in the ATP binding site of the PDK1 kinase domain. The NF-KB response-element reporter construct was conjugated to a luciferase reporter gene. The entry plasmids (pENTRl A) 24 and the destination plasmids (pLenti4/V5-DEST) were kind gifts from Dr. Paul Rennie (The Prostate Centre, Vancouver, Canada). 2.2 METHODS 2.2.1 Cell Fractionation Cell fractionation was described before [159]. Briefly, cells were washed twice with ice cold PBS, resuspended in hypotonic HEPES buffer (10 mM HEPES pH 7.9, 5 mM MgCh, 40 mM KC1) and left on ice for 30 min, aspirated repeatedly through a 25-gauge needle (30 strokes), and centrifuged at 200xg at 4°C for 10 min to pellet nuclei. The supernatant was centrifuged at 50,000 rpm for 90 min at 4°C to pellet membranes, and the resulting remaining supernatant represented the cytosol. 2.2.2 Griess Assay The Sulfanilamide Solution (1% sulphanilamide in 5% phosphoric acid) and NED solution (0.1% N-l-napthylethylenediamine dihydrocholride in water) are allowed to equilibrate to room temperature. Add 50 uL of each experimental sample to wells in duplicate. 50 uL of Sulfanilamide Solution was dispensed to all wells. The 96-well plate was incubated in dark for 5 min at room temperatue. 50 uL of the NED Solution was then added to the wells. The plate was incubated again for 5 min in dark. The absorbance was measured within 30 min in a plate reader with a filter at 535 nm. 2.2.3 Immunoprecipitation 400 ug protein lysate was made up to 200 ul and precleared with 20 pi protein A/G sepharose slurry (Sigma, Germany) at 4°C for 10 min. The supernatant was transferred to fresh tubes after centrifuged at 14,000 rpm for 10 min. 5 ul of the appropriate antibody was added, and the mix was rotated overnight at 4°C. 50 ul of 25 protein A/G slurry was added to the tubes the next day followed by another hour rotation at 4°C. The beads were collected by pulse centrifugation and resuspended in PBS after three washes. The beads were then heated in 90°C for 5 min to dissociate the immunocomplexes. The supernatant was transferred to clean tubes and separated on SDS-PAGE. 2.2.4 PDK1 Kinase Assay 5 pi MgC12 (lOOiruM), 10 pi of ADB, 5 pi of PDK tide and 10 ul of ATP were added to the beads from immunoprecipitation. The mix was incubated in 30°C for 20 min. 15 pi of the solution was taken off and spotted onto P81 paper. The P81 paper was placed into scintillation fluid after washing in 0.01% phosphoric acid 10 min for 6 times and counted. 2.2.5 Immunoblotting Cells were washed twice with ice-cold PBS, resuspended in homogenization buffer for 15 min, sonicated for three 5 s intervals on ice at 30% output, before being centrifuged at 13,000 rpm for 10 min. The protein concentration in the supernatant was determined by the Bradford assay. 60-70 ug of protein from each sample was resolved using 10% SDS-PAGE before transferring to nitrocellulose membranes (Bio-Rad). The blots were blocked in 5% skim milk in TBS-T (20mM Tris-HCl pH 7.4, 250 mM NaCl, 0.05% Tween-20) for 1 hr before probing with the appropriate primary antibody for 2 hr at room temperature or over night at 4°C. The blots were washed with TBS-T for 5 min three times, before being incubated with appropriate secondary antibody for 1 hr. Following 3 further washes in TBS-T, they were developed using the enhanced chemiluminescence detection system (ECL, Amersham, Montreal, Quebec). Phospho-PKB, phospho-p65, phospho-p70S6K, phospho-PDKl, phospho-MAPK were purchased from Cell Signaling (Missisaugua, Ontario). Antibodies to PKB kinase (PDK1), p65, DcBalpha, iNOS were purchased from Santa 26 Cruz Biotechnologies (Santa Cruz, CA). 2.2.6 Nuclear Preparation Cells were washed once with ice cold PBS, resuspended in 200 uL Buffer A (10 mM HEPES pH 7.9, 10 mM KC1, 0.1 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 0.5 mM PMSF) for 10 min. The cell suspension was vortexed for 10 s before being spun at 1,000 rpm for 30 s. The supernatant was removed and the pellet was centrifuged again at 12,000 rpm for 5min. The supernatant was removed and the nuclear pellet resuspended in 30 uL of Buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, ImM EDTA, ImM EGTA, 25% glycerol, 1 mM DTT, 1 mM PMSF). The sample was vigorously rocked for 15 min and subsequently centrifuged for 5 min at 12,000 rpm. The supernatant was retained and the protein concentration determined by the Bradford assay. Samples were stored at -80°C until use. 2.2.7 Electrophoretic Mobility Shift Assay (EMS A) A synthetic kB oligonucleotide was cloned into the doing vector, pBS (Stratagene, La Jolla, CA), using the EcoRl and Hindlll sites, to create pBS-EMSAicB. To radiolabel the probe, it was excised from pBS-EMSAkB using EcoRl and Hindlll, and labelled using [y- P]dCTP (Amersham, Montreal, Quebec, Canada) and the Klenow fragment of DNA polymerase (New England Biolabs, Missisauga, Ontario, Canda). The probe was then purified by running on a 5% non-denaturing gel, cutting out the fragment, and incubating the gel slice in elution buffter [(0.6M ammonium acetate, ImM EDTA, 0.1% sodium dodecyl sulphate (SDS)] overnight. lOug of nuclear extract were preincubated in binding buffer (20 mM Hepes pH 7.9, 100 mM KC1, 10% glycerol, 1 mM DTT) and 1 ug of poly dldC (Amersham, Montreal, Quebec), for 15 min. 20,000 CPM of hot probe was then added, and the reaction mixture incubated at room temperature for 30 min, and subsequently resolved 27 on a 5% non-denaturing polyacrylamide gel in 0.25xTBE at 200V for 1.5 hr. The gel was then dried for 45 min before phosphoimging analysis using a Bio-Rad molecular imager FX. 2.2.8 Transient Transfection Raw 264.7 cells (9xl05/well) were seeded into the 6-well plate. The next day cells were about 50-60% confluent, and transfection was carried out using Superfect Transfection Reagent (Qiagen) following supplier's instructions at a ratio of 1:5 (DNA ug : Superfect) with lug plasmid DNA per well. Cells were incubated for 20 hr, and then fresh medium containing 10% heat-inactivated FBS was added after one wash. 2.2.9 Luciferase Assay 0.2 ug of an NF-icB-dependent reporter containing 4 repeats of the KB consensus sequence was co-transfected to Raw 264.7 cells as described above. Cells were lysed with Glyo Lysis Buffer (Promega) and assayed for luciferase activity using Luciferase Assay reagent (Promega). 2.2.10 Lentiviral Vector Construction The KD PDK1 plasmids were used to construct the lentiviral vectors. The oligonucleotides were annealed and then ligated into the pENTRl A entry plasmid via Kpnl/Xmal sites. DB3.1 E.coli (Invitrogen) were transformed, and clones were tested on agarose gel. The construction of the packaging vector pCMV A R8.2, and VSV envelope vector pMD.G have been described before. The entry clones were transposed to the pLenti4/V5-DEST destination plasmids by Gateway LR Clonase Enzyme Mix (Invitrogen). (Fig. 4A) Positive clones were isolated. All plasmids preparation were carried out using Qiagen Miniprep or Midiprep kits, and plasmid 28 purifications were performed using a Qiagen Plasmid kit. 2.2.11 Lentivirus Packaging The packaging cell line 293T HEK (5xl06) were plated in 10-cm tissue culture dish the night before transfection. Three hours prior to transfection, the medium was removed from the cells and replaced with fresh growth medium. 10 ug transducing vector, 7.5 ug packaging vector pCMV A R8.2, and 2.5 ug VSV envelope pMD.G were co-transfected by 250 pi calcium phosphate (0.5M CaCh). A vector carrying a GFP gene was used as positive control. The medium was changed 20 hr later. GFP fluorescent positive cells were observed under fluorescent microscopy. Conditioned medium was then collected and cleared of debris by filtered through a 0.45 um filter and stored at -80 °C. This collection was repeated the next day, and media from the 2 days were pooled and ultra-centrifuged at 25,000 rpm at 4 °C for 100 min. The pellet was resuspended in 75 pi medium (overnight at 4°C), and aliquots were stored at -80°C. 2.2.12 Zeocin Selection of Raw 264.7 cells Raw 264.7 cells were seeded to 10-cm plates and cultured to about 80% confluent. The medium was then changed to different concentration zeocin medium (0, 100, 250, 400, 600, 800, 1000 ug/ml). Unlike other antibiotics, zeocin sensitive cells don't round up and detach from plate. Instead, as shown in Fig. 5, on day 3, cells started to show some characteristic morphological changes: vast increase in size, and the appearance of long appendages. On day 8, more changes can be observed including presence of large empty vesicles in cytoplasm, cell debris after breaking down. At the end of two weeks, cells in the plate all die in concentration as low as 600ug/ml. 29 2.2.13 Transduction of Target Cells Raw 264.7 cells (3xl05) were seeded to the 12-well plate and cultured with the lentiviruses the next day for 24 hr, and then the culture medium was replaced. After 24 hr, the medium with 600 ug/ml zoecin was added. Cells underwent antibiotics selection for 2 weeks and the remaining colonies are pooled and kept growing in zeocin-containing medium. 30 K61Q Kpnl Xmal _ l pCMV5-Myc Ligate annealed olisos OLIGO _pENTRlA Clonase Reaction • CMV pLenti4/V 5-DEST 'AWMM mmm-> CMV OLIGO pPDKl-KD-LV co-transfection pCMVAR8.2 pMD.G Figure 4A. Lentiviral vector construction HEK 293T Packaging of vector RNAs, assembling and budding of progeny virus !> _ Harvest conditioned media every 24 hrs V for 3 days • Figure 4B. Lentivral packaging 31 Day 1 normal cell morphology Day 3 vast increase in cell size appearance of appendage Day 8 presence of large empty vesicles in cytoplasm cell debris Figure 5. Zeocin selection of Raw 264.7 cells 32 CHAPTER 3 -RATIONALE AND HYPOTHESIS As discussed before, PI-3K is involved in the LPS signaling in macrophages, although there is paradox about their interaction probably due to cell model variability. NO production is completely blocked by PI3-K inhibitors wortmannin and LY 294002 from LPS-induced bone marrow-derived macrophages [111]. In murine macrophages Raw 264.7, the PI3-K homologues mTOR/FRAP have been implicated in the phosphorylation and activation of iNOS [161]. What's more, integrin-linked kinase (ILK), which couples integrins and growth factors to downsteam signaling pathways, has been shown to regulate iNOS expression and NO production in murine macrophages in an NF-icB-dependent manner [162]. ILK regulates NF-KB activity through PKB/Akt by phosphorylating Ser473 in a PI3-K-dependent manner [114-117,162]. PDK1 as one of the important downstream of PI3-K and the other important upstream kinase of PKB/Akt, its role in LPS signaling in macrophages has not been investigated before. In this study, we hypothesized that PDK1 regulates iNOS and consequently nitric oxide production in macrophages activated by LPS. Pl.-k II .K I i n : OK l ' K H m f 1 N O Figure 6. Hypothesis. 33 CHAPTER 4 - PDK1 inhibition by TPCK 4.1 RATIONALE To investigate the role of PDK1 in LPS signaling in Raw 264.7 cells, we first tried to inhibit PDK1 signaling by a pharmacological inhibitor. N-a-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has been shown to be an inhibitor of PDK-1 signaling [163]. So we treated cells with TPCK before LPS stimulation and examined the signaling events and used iNOS protein expression and NO production as readouts. 4.2 RESULTS 4.2.1 PDK1 activation by LPS stimulation in murine macrophages First of all, we confirmed that PDK1 is activated after LPS stimulation in Raw 264.7 cells. As discussed before, PDK1 relocates from the cytosol to the plasma membrane in stimulated cells and thus facilitates subsequent PKB/Akt activation [107, 108]. Raw cells were treated with TPCK before stimulated with LPS for indicated time point. Cell pellets were fractionated and the membrane fraction was analyzed. As shown in Fig. 7A, PDK1 relocated to the membrane fraction in a time-dependent manner. We then examined the PDK1 kinase activity in the membrane fraction. PDK1 activity increased rapidly after LPS stimulation; reaching its peak at 3 min (Fig. 7B). We also did the kinase assay in a longer time course till 60 min (data not shown), which showed a similar change corresponding to the western blotting results. However, this increase of catalytic activity may be independent of PDK1 relocation as other study showed that a catalytically inactive kinase mutant of PDK1 was still capable of translocating[108]. 34 42.2 TPCK inhibition of iNOS expression and NO production To investigate the involvement of PDK1 in Raw 264.7 cells NO production, we examined iNOS protein expression and NO production by inhibiting PDK1 with TPCK. The cells were treated with TPCK, followed by measurement of iNOS and NO with western blotting and Griess assay respectively. When cells were treated with TPCK (5, 10, 25, 50pM) prior to LPS stimulation for 18 hrs, iNOS expression was suppressed in a concentration-dependent manner (Fig. 8A). Similarly, Raw 264.7 macrophages consistently produced NO at least in 72 hr following LPS challenge, while TPCK inhibited the NO production concentration-dependently (Fig. 8B). These data confirmed that TPCK could reduce NO production in Raw 264.7 cells in response to LPS. 4.2.3 TPCK inhibition of PDK1 activation Previous study has shown that pharmacological inhibitor TPCK may not be acting via direct inhibition of the kinase activity of PDK1 or it its kinase substrates but may be preventing the PDKl-substrate interaction inside the cell [163]. Therefore, we examined the phosphorylation of the PDKl downstream proteins to address PDKl inhibition by TPCK. As mentioned previously, PKB/Akt and p70 S6K are well-defined downstream targets of PDKl activation. As shown in Fig. 9A, PKB/Akt phosphorylation at Thr308 site was inhibited by treating the cells with TPCK prior to stimulation. What's more, Ser473 site phosphorylation was also suppressed by TPCK treatment to a lesser extent. p70 S6K phosphorylation at Ser389 site showed similar results. Meanwhile, the MAPK pathway was not affected. (Fig. 9B) In addition, phosphorylation of GSK-3p at Ser9 and S6 ribosomal protein at Ser240/244 also showed similar suppression by TPCK treatment (data not shown). These blots confirmed the PDKl signaling inhibition by TPCK while this inhibition does not impact the MAPK pathway which could also induce NO production. 35 4.2.4 TPCK inhibition of NF-KB pathway iNOS expression is induced by the transcription factor N F - K B . The present study demonstrated the effects on NF-KB activity in TPCK treated Raw 264.7 macrophages. We transiently transfected the Raw cells with the NF-KB-based luciferase reporter as described in Materials and Methods. LPS stimulation increased N F - K B activity up to five folds, while it decreased to basal level by TPCK inhibition (5, 10, 25, 50uM) (Fig. 10A). Furthermore, as the NF-KB activation is preceded by IKB-CX degradation, we also examined the IkB-a level in cells after LPS stimulation. We showed that IKB-CI degrades as early as in 10 min and is totally gone in 15 min after LPS stimulation. While TPCK at 20uM significantly delayed this process; when at 50uM, the degradation was completely abrogated (Fig. 10B). 4.2.5 TPCK inhibition of NF-KB nuclear binding To further confirm our findings, we also examined the NF-KB nuclear binding in Raw cells. As shown in Fig. 11, the p65 nuclear binding increased after LPS stimulation in Raw 264.7 cells. However, pre-treatment of TPCK greatly inhibited the event. Taken together, the data indicates that PDK inhibition by TPCK decreases iNOS induction and NO production which is correlated with the attenuation of NF-KB activity in LPS stimulated Raw 264.7 cells. 36 B NO assay 60 1 50 B 40 O 30 s 20 O z 10 0 / 24 hr 48 hr i I i i i • i III V I I 72 hr i l Figure 8. TPCK inhibition of iNOS expression and NO production. (A) The cells were stimulated with LPS (lOOng/ml) for 18 hr in the absence or presence of TPCK at the designated concentration (uM). (B) Raw 264.7 macrophages were stimulated with LPS in the absence or presence of TPCK at the designated concentration. Medium was collected at 24 hr, 48 hr and 72 hr respectively. The results are representative of at least three independent experiments. 38 B P85 p70 CTL LPS LPS+TPCK (uM) IP:Akt 5 10 25 t^lfP '^t^^^Kk' ffi: Phospho-Akt (Thr308) tf& m* D3: Phospho-Akt (Ser473) IB:Aktl/2 densitometry analysis 2.5 so . i-E 1.8 0.5 CBfc • Thr308 • Ser473 CTL LPS TPCK 5 TPCK 10 TPCK 25 CTL LPS LPS+TPCK (uM) 5 10 25 50 Phospho-p70S6 Kinase (Ser389) Phospho-MAPK (p42/44) GAPDH Figure 9. TPCK inhibition of PDK1 activation. (A) Raw 264.7 cells were pretreated with TPCK at the indicated concentrations and then stimulated with LPS for 20 min. PKB was immunoprecipitated from equivalent amounts of cell lysate and after resolving the samples on 10%SDS-PAGE, and transferring to membranes, the samples were probed with the antibodies indicated. (B) Sample lysates were prepared as in A, except that the westerns were probed directly with the antibodies indicated. 39 A NF-kB based luciferase assay T P C K (pM) B LPS (lOOng/ml) 10 15 20 30 (min) T P C K 0 (uM) T P C K 20 T P C K 50 I K B - C X G A P D H Figure 10. TPCK inhibition of NF-KB pathway. (A) The data indicates the fold change of NF-KB acitivity. Raw 264.7 cells were transfected with the NF-KB-based luciferase plasmids as described in Materials and Methods. (B) Raw 264.7 macrophages were challenged by LPS at the presence or absence of TPCK (uM) for designated time. IKB-O degradation was greatly suppressed by TPCK at 20 uM after LPS stimulation, and completely abrogated at 50uM. 40 1 2 3 4 5 6 Figure 11. T P C K inhibition of NF-KB nuclear binding. p65 D N A binding was carried out using E M S A , as previously described. Raw 264.7 cells were treated with L P S for 60 min after exposure to T P C K . (1) control; (2) L P S lOOng/ml; (3-6) T P C K 5, 10, 25, 50ug/ml. 41 CHAPTER 5 - PDK1 inhibition by transient transfection 5.1 RATIONALE In order to exclude the possibility that pharmacological inhibitors could have non-specific effects in cells, PDK1 was inhibited by a kinase-dead PDK1 construct transiently transfected into the cells. 5.2 RESULTS 5.2.1 iNOS induction and NO production in transfected Raw cells Cells were transiently transfected with the empty vector (EV) or PDK1 kinase-dead (KD) construct. Transfected cells were replaced back to complete growth medium with 10% serum 20 hr after transfection. LPS treatment was performed the next day. iNOS expression was greatly reduced in PDK1 KD expressing cells compared to the EV transfected cells (Fig. 12A); NO production was down-regulated to basal level too (Fig. 12B). 5.2.2 PDK1 signaling in transfected Raw cells Similarly, we examined the PDK1 signaling in the transfected macrophages. As shown in Fig. 13, phosphorylation of PKB/Akt at Thr308 site, the downstream target of PDK1, was suppressed in PDK1 KD expressing cells compared to the EV transfected Raw 264.7 cells. However, the signals were quite weak compared to other experiments, this may due to the low volume of phospho-PKB/Akt at Thr308 in the cell lysate. 42 5.2.3 NF-KB pathway in transfected Raw cells We next examined the NF-KB pathway in the transfected macrophages. As LPS has been found to induce phosphorylation of the p65 at Ser536 [164], we detected the phosphorylated form of p65 on Ser536 in the cell lysate using the specific Ab. Similar to the PKB/Akt result, phosphorylation of p65 at Ser536 site was also inhibited. (Fig. 14A) What's more, LPS treatment of Raw 264.7 cells elicited about four-fold increase of NF-KB activity in EV transfected cells, yet it was greatly suppressed in PDKl KD expressing cells (Fig. 14B). Taken together, these results strengthen the previous finding using pharmacological inhibitor showing that PDKl kinase activity is required for NO production in Raw 264.7 cells in that the partial suppression of PDKl kinase activity leads to reduced iNOS expression and NO production. 43 A EV EV KD KD +LPS +LPS B iNOS GAPDH Figure 12. iNOS induction and NO production in transfected Raw cells. Raw 264.7 macrophages were transiently transfected with empty vector (EV) or PDK1 kinase-dead (KD) plasmid. (A) Cells were treated with LPS (lOOng/ml) for 18 hr before harvested. (B) Medium was collected 48 hr after LPS challenge. •i-i Figure 13. PDK1 signaling in transfected Raw 264.7 cells. Transfected Raw cells were treated with LPS 24 hr after changed back to 10% FBS containing medium for 10 min. 90 ug proteins were subjected to immunoblotting and probed with phospho-Akt (Thr308) antibody overnight at 4°C. The data shows the Thr308 site phosphorylation on PKB/Akt is inhibited when cells were transiently transfected with PDK1 KD and stimulated with LPS. 45 B NF-kB based luciferase assay Figure 14. N F - K B pathway in transfected Raw cells. (A) The panels show the Ser536 site on p65 is inhibited when cells were transiently transfected with PDK1 KD and stimulated with LPS. (B) Raw 264.7 cells were transiently co-transfected with NF-xB-based luciferase plasmid and empty vector (EV) or PDK1 kinase-dead (KD) construct. This intervention results in the inhibition of LPS-mediated N F - K B reporter activity. •16 CHAPTER 6 - P D K l inhibition by stable transduction 6.1 RATIONALE Due to the fact that the transfection efficiency for macrophages is quite low, we performed a stable transfection to further strengthen our findings. The lentiviral vector is an ideal tool. Lentiviral vectors are retroviral vectors based on HTV-1. They can stably integrate into the host cell genome, expressing the target gene product. With the development of vector design, lentiviral vectors have been widely applied to many areas of medical research, becoming a strong and promising gene transfer tool for investigation and gene therapy. In the present work, we made a lentiviral vector expressing kinase-dead PDKl. 6.2 RESULTS 6.2.1 NO production in transduced Raw cells Cells were transduced with PDKl KD-LV and selected by 600 u,g/ml zeocin. The pooled colonies were kept growing in zeocin-containing medium. There was about 50% reduction of NO production in transduced cells expressing KD PDKl compared to normal Raw 264.7 cells. (Fig. 15) In addition, we also made a lentiviral vector expressing wild-type (WT) PDKl. However, the NO production was the same in PDKl WT-LV-transduced cells and regular Raw cells, which was consistent with our previous finding. This also excluded the possibility that the NO reduction is due to the vector system itself. 6.2.2 PDKl signaling in transduced Raw cells Next we performed experiments to examine the PDKl signaling in those transduced 47 Raw cells. There was no increase of phosphorylated Akt at Thr308 after LPS stimulation in KD-LV-transduced cells compared to regular Raw 264.7 cells (Fig. 16A). It is worth mentioning that there is basal increase in phospho-PKB/Akt signals in transduced cells. This may be due to the auto-compensation in cells when one protein is being suppressed persistently. Similar results were seen in PDKl and phosphorylated PDKl at Ser241 protein expression (Fig. 16B). 6.2.3 NF-KB pathway in transduced Raw cells Again, similar experiments were done to study the NF-KB pathway in those transduced Raw cells. The phosphorylation of p65 at Ser536 is attenuated in the KD-LV-transduced cells. (Fig. 17A) Furthermore, IKB-CI degradation is also significantly inhibited in KD-LV-transduced cells compared to Fig. 8B. (Fig. 17B) Taken together, the lentiviral data again confirmed our finding that PDKl is involved in the signaling pathway of NO production in Raw 264.7 cells stimulated by LPS. Nevertheless, one has to admit that the results in LV-transduced cells are just as the same extent of change as, if not less than, those in transfected cells. The reason for this is likely that PDKl is a universal protein in cells involved in a variety of activities including cell survival, cell growth etc. When PDKl is persistently suppressed in cell, the whole cell physiology is impaired; this is consistent with our finding that the transduced cells show certain changes in morphology as well as growth rate. These changes may contribute to the less significant response to LPS stimulation. 48 NO assay 35 i 30 1 regcont. regLPS KD-LV KD-LVLPS com. Figure 15. NO production in transduced Raw cells. The histogram shows that NO produced is about fifty percents attenuated in the PDK1 KD-vector transduced cells. The medium was analyzed after cells exposure to LPS for 24 hr. The results are representative of three independent experiments. 49 A REG REG KD-LV KD-LV +LPS +LPS (Thr308) Figure 16. P D K l signaling in transduced Raw cells. Immunoblotting was performed to examine the phosphorylation status of PKB/Akt at Thr308 site (A) and PDKl at Ser241 site (B) in the PDKl KD-vector transduced Raw cells compared to the normal Raw cells. 50 Figure 17. NF-KB pathway in transduced Raw cells. (A) The upper panels show the phosphorylation status of p65 at Ser536 site. The lower lower panel was the densitometry analysis of the blots. (B) The PDK1 KD-LV transduced Raw cells were stimulated with LPS(100ng/ml) for indicated time. IicB-a degradation was examined by imrnunoblotting. 51 CHAPTER 7 - CONCLUSION The body of work presented here has examined the role of PDKl in the LPS-induced signaling pathway of iNOS induction and NO production in a murine macrophage cell line, Raw 264.7 cells. LPS, upon binding to the TLR4, CD14, MD2 receptor complex on the cell surface, activated PDKl resulting in PDKl relocation from cytosol to membrane. N F - K B , which is required for the synthesis of many inflammatory mediators, including NO, was found to be a point of convergence of many of these pathways. Degradation of hcB-a is required for the activation of NF-KB dimer. TPCK inhibits PDKl downstream signaling, but does not impact on MAPK signaling. TPCK decreases iNOS induction, subsequently NO production through NF-KB inhibition via at least the attenuation of IKB-CC degradation. In order to strengthen the data using pharmacological means, we also inhibited PDKl kinase activity in the cells by two other ways. Both transient transfection of the PDKl KD plamids and stable transduction of the lentiviral vectors expressing KD PDKl showed that PDKl activity is required for LPS-induced iNOS induction and NO production. In addition, PDKl is involed directly or indirectly in the LPS-induced phosphorylation of p65. However, it is worth mentioning that when we overexpressed PDKl by transfecting with the PDKl wild type construct, we did not see a dramatic increase in iNOS or NO production. It might be possible that endogenous PDKl is enough to drive the pathway. PDKl/Akt pathway has been extensively studied in cell survival regulation and cancer progress. Recent studies discovered that PDKl also plays a critical role in adaptive immune system. Heather et al. [165] found that complete PDKl loss blocked T cell differentiation in the thymus, whereas reduced PDKl expression allowed T cell differentiation but blocked proliferative expansion. 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