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Regulation of human monocyte functional properties by phosphatidylinositide 3-kinase Lee, Jimmy Sheng-I 2006

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Regulation of Human Monocyte Functional Properties by Phosphatidylinositide 3-Kinase by Jimmy Sheng-I Lee B.Sc. McGi l l University, 1998 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF COMBINED DOCTOR OF PHILOSOPHY A N D DOCTOR OF MEDICINE in THE F A C U L T Y OF G R A D U A T E STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH C O L U M B I A April 2006 © Jimmy Sheng-I Lee, 2006 ABSTRACT Mononuclear phagocytes are important regulators and effectors of both the innate and acquired immune responses. Extensive research has highlighted an important role for phosphoinositides in monocyte cell regulation. The 3'-phosphoinositide metabolites, produced by phosphatidylinositide 3-kinase (PI3K) family of lipid kinases are known to be involved in regulating numerous monocyte activities including phagocytosis, adherence, oxidative burst, and cytokine secretion. An important research objective is to develop an understanding of how specificity is achieved in PI3K signaling for these diverse biological functions. The goal of this thesis was to determine when particular monocyte functional properties are governed by PI3K in an isoform-specific manner versus situations in which PI3K family members have redundant functions. Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p i 10a isoform of PI3K was silenced using a lentiviral vector expressing short hairpin RNA. This resulted in the generation of stable THP-1 and U-937 human monocytic cell lines deficient in p i 10a. The role of p i 10a in regulating cell adherence, phagocytosis, the phagocyte oxidative burst, and LPS-induced cytokine secretion was examined. Monocyte adherence induced in response to either LPS or vitamin D 3 was blocked by PI3K inhibitor LY294002. However, while adherence induced in response to D3 was i i sensitive to silencing of p i 10a, LPS-induced adherence was not. These findings demonstrate that LPS and vitamin D 3 use distinct isoforms of class IA-PI3K to induce functional responses. We also observed that p i 10a was required for phagocytosis of IgG and serum opsonized particles in differentiated U-937 cells. The phagocyte oxidative burst induced in response to either P M A or opsonized zymosan in differentiated THP-1 cells was also found to be dependent on p i 10a. Furthermore, p i 10a was observed to exert selective and bi-directional effects on the secretion of pivotal cytokines apparently independently of other PI3K isoforms. LPS stimulation of p i 10a deficient THP-1 cells demonstrated that p i 10a was required for inducing IL-12 and IL-6 production, whereas this isoform of PI3K appeared to negatively regulate the production of TNF-a and IL-10. The results reported in this thesis demonstrate that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology. Furthermore, taken together, the data suggest that p i 10a PI3K is involved in regulating important monocyte effector functions independently of other PI3K family members. 111 TABLE OF CONTENTS ABSTRACT.. i i TABLE OF CONTENT iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xii ACKNOWLEDGMENTS xv CHAPTER I: INTRODUCTION 1 1.1 Mononuclear phagocytes 1 1.1.1 Macrophage origin, morphology, and heterogeneity 1 1.1.2 Macrophages respond to heterogeneous stimuli through a group of diverse cell surface receptors 5 1.1.3 Activation of macrophages 12 Functional properties of macrophages 13 1.2 Phosphatidylinositide 3-kinase 17 1.2.1 The PI3K family and 3'-phosphoinositides 17 1.2.2 Activation and regulation of PI3K 26 1.2.3 PI3K Effectors .. 40 1.2.4 Negative regulation of PI3K products: Phosphatases 44 1.2.5 Multiple isoforms of PI3K confer diversity in regulation of cellular processes ; 45 1.3 R E S E A R C H OBJECTIVES 49 iv 1.3.1 Objective 1: To selectively downregulate a distinct PI3K isoform using lentiviral-delivered siRNA '. 50 1.3.2 Objective 2: Examining the role of p i 10a isoform in monocyte adherence, FcyR- and CR3-mediated phagocytosis 52 1.3.3 Objective 3: Role of pi 10a isoform in LPS-induced cytokine secretion and in oxidative burst 54 CHAPTER II: MATERIAL AND METHODS 56 2.1 Reagents, chemicals, and cell lines 56 2.2 Monocyte transfection and electroporation with oligonucleotides and siRNA.... 57 2.3 Lentiviral preparation and transduction of monocytic cells 58 2.4 Western blot analysis 62 2.5 Adherence assays 62 2.6 Dual luciferase assays ..... 63 2.7 Flow cytometry analysis 63 2.8 Cytokine Measurement 64 2.9 RT-PCR :. 64 2.10 Phagocytosis assay 65 2.11 Superoxide assay 66 2.12 Statistical analysis 66 CHAPTER III: LENTIVIRAL-MEDIATED DELIVERY OF siRNA INTO HUMAN MONOCYTIC CELL LINES 67 3.1 Introduction 67 3.2 Transfection of anti-sense oligonucleotides and siRNAs into monocytic cell lines using cationic lipids and electroporation 68 3.3 Lentiviral-fnediated delivery of siRNA 72 3.4 Silencing of PI3R p i 10a isoform in human monocytic cell lines 80 •3.5 Transduced H E K 293T cells can be further transfected with reporter plasmids.. 80 3.6 Discussion 83 C H A P T E R IV: R O L E O F p i 10a PI3K IN R E G U L A T I N G M O N O C Y T E A D H E R E N C E AND P H A G O C Y T O S I S 87 4.1 LPS and vitamin D 3 induced adherence : 87 4.1.1 Monocyte adherence induced by D 3 , but not LPS is dependent on p i 10a. .. 88 4.1.2 CD1 lb expression in response to vitamin D 3 : 92 4.2 Regulation of phagocytosis by p i 10a PI3K. 95 4.2.1 Effect of p i 10a silencing on phagocytosis ....98 4.3 Discussion ; .„..•;• 103 C H A P T E R V: R O L E O F PI3K pl lOa IN R E G U L A T I N G C Y T O K I N E PRODUCTION AND T H E O X I D A T I V E BURST 110 5.1 Toll-like Receptor family 110 5.2 PI3K and expression of pro-inflammatory cytokines in TLR4 signaling 123 5.3 Effects of p i 10a silencing on LPS-induced cytokine expression 126 5.4 Activation of signaling pathways by p i 10a in response to LPS 142 5.4.1 Western blot analysis ....142 5.4.2 mRNA levels for TNF-a, IL-6, IL-10, and IL-12 146 5.5 NADPH-dependent oxidase and PI3K 148 vi 5.5.1 Assembly of the NADPH-dependent oxidase complex is highly regulated and requires PI3K activity 148 5.5.2 Effect of p i 10a silencing on activation of the phagocyte oxidase by P M A and opsonized zymosan 156 5.6 Discussion : '. 159 CHAPTER VI: DISCUSSION 180 6.1 Choice of gene silencing strategy may affect phenotype... 180 6.2 A single receptor complex can activate multiple PI3K isoforms, and different receptors can lead to the same biologic response by using different PI3K isoforms . 183 6.3 Control of diverse monocyte effector functions by a single isoform of PI3K.... 184 6.4 Perspectives and significance 187 REFERENCES 189 vn L I S T O F T A B L E S Table 1-1. Overview of macrophage receptors in immune recognition 9 Table 1-2. Toll-like receptors arid their ligands 11 Table 1-3. P I3K family in eukaryotes 25 Table 1-4. Phenotypes of targeted regulatory and catalytic subunits of P I3K 48 Table 2-1. Short Hairpin R N A encoding sequences targeting human p l l O a m R N A . 60 Table 2-2. Primer sequences and conditions for R T - P C R 65 viii LIST OF FIGURES Figure 1-1. Schematic diagram showing differentiation, distribution, and activation of macrophages and multinucleated giant cells. ...2 Figure 1-2. Toll-like receptor family and their principle ligands 10 Figure 1-3. Structure of phosphoinositides and pathways of 3-phosphoinositide synthesis and degradation 21 Figure 1-4. Mammalian PI3K catalytic and regulatory subunits 22 Figure 1-5. Regulation of class IA PI3K 36 Figure 1-6. Control of insulin and PI3K signaling by mTOR and S6K1 38 Figure 1-7. PI3K signal transduction pathways showing interaction of various 3'-PI with PH or FYVE domain-containing proteins 43 Figure 3-1. Transfection of antisense oligonucleotide into THP-1 cells by Oligofectamine 71 Figure 3-2. Construction of a lentiviral vector for transduction of shRNA into target cells 74 Figure 3-3. Transduction of monocytic cell lines by lentiviral vectors is efficient and generates stable cell lines deficient in pllOoc 79 Figure 3-4. Silencing of an exogenous gene in HEK 293T cells expressing shRNA. 82 Figure 4-1. Monocyte adherence induced by D3, but not LPS is dependent on pllOa. 91 Figure 4-2. CDllb induction by D3 is attenuated in pllOa deficient THP-1 cells.. 93 ix Figure 4-3. Surface receptor expression, opsonization of latex beads, and phagocytosis of IgG and serum opsonized latex beads by PMA-differentiated U-937 cells deficient in PI3K pllOa subunit 101 Figure 5-1. Schematic diagram of TLR4 signaling pathways 119 Figure 5-2. Model of PI3K involvement in TLR4-TRIF pathway and PI3K-dependent IRF3 activation in TLR3 signaling 121 Figure 5-3. Results of ELISA for TNF-a, IL-6, IL-10, and IL-12p40 at 5h and 18h post LPS-stimulation of THP-1 cells 131 Figure 5-4. Effect of neutralizing antibodies to TLR-4 and TLR-2 on LPS and zymosan induced IL-12p40 and IL-6 production in control shRNA transduced THP-1 cells. 134 Figure 5-5. Effect of neutralizing anti-TNF-a or anti-IL-10 antibodies on LPS-induced IL-12p40 and IL-6 production by THP-1 cells 138 Figure 5-6. Coincubation of neutralizing anti-TNF-a and anti-IL-10 antibodies did not restore LPS-induced IL-12p40 and IL-6 production. 141 Figure 5-7. Western blot analysis 144 Figure 5-8. RT-PCR for TNF-a, IL-12p40, IL-6, and IL-10, mRNA in LPS-stimulated THP-1 cells deficient in PI3K pi 10a 147 Figure 5-9. Model of NADPH oxidase assembly and activation 153 Figure 5-10. p85/pll0a PI3K is required for PMA- and opsonized zymosan-induced oxidase activation 157 Figure 5-11. Model of PI3K in LPS-induced monocyte signaling 169 x F i g u r e 6-1. M o d e l o f P I 3 K i s o f o r m usage l i n k e d to m o n o c y t e r e c e p t o r s a n d f u n c t i o n a l responses xi LIST O F ABBREVIATIONS A M P K AMP-activated protein kinase AP-1 Activator protein-1 A R E AU-rich elements ARF6 ADP ribosylation factor 6 ARNO A R F nucleotide binding site opener ASK-1 Apoptosis signal-regulating kinase 1 ATF-2 Activating transcription factor 2 B T K Bruton tyrosine kinase cAMP Cyclic A M P CBP CREB binding protein CGD Chronic granulomatous disease CR3 Complement receptor 3 CREB Cyclic A M P response element binding protein D 3 l a , 25-dihydroxycholecalciferol D A G Diacylglycerol D N Dominant negative EGFR Epidermal growth factor receptor E R K Extracellular signal-regulated kinase FcyR Fey receptor FGD1 Faciogenital dysplasia Gab Grb2-associated binder GAP GTPase-activating proteins GEF Guanine nucleotide exchange factors GRP General receptor for phosphoinositides Hrs Hepatocyte growth factor regulated tyrosine'kinase substrate I K B Inhibitor of nuclear factor-KB I K K Inhibitor of nuclear factor-KB-kinase complex IL Interleukin IPs Inositol-1,4,5-trisphosphate IRAK IL-1 receptor associated kinase IRF Interferon regulatory factor ISRE Interferon-stimulated response elements ITK Inducible T-cell kinase JIP JNK-interactirtg protein JNK Jun N-terminal kinase K O Knockout LPS Lipopolysaccharide LTR Long terminal repeats M A L MyD88-adaptor like protein M A P K Mitogen-activated protein kinase M A P K A P - K 2 MAP-kinase-activated protein kinase-2 M A P K K K M A P kinase kinase kinase M E K K M A P K / E R K kinase kinase M K 2 MAP-kinase-activated protein kinase-2 xii MOI Multiplicity of infection MPO Myeloperoxidase MPS Mononuclear'phagocyte system mTOR Mammalian target of rapamycin MyD88 Myeloid differentiation primary-response protein 88 N A D P H ' Nicotinamide adenine dinucleotide phosphate, reduced NIK N F K B inducing kinase NOD Nucleotide-binding oligomerization domain OPZ Opsonized zymosan P A K p21 activated kinase PDE3B Phosphodiesterase 3B PDK 3 '-phosphoinositide-dependent kinase PH Pleckstrin homology PHLPP PH domain leucine-rich repeat protein phosphatase PI Phosphoinositides PI3K Phosphoinositide 3-kinase, P K A Protein kinases A PKB Protein kinases B PKC Protein kinases C P M A Phorbol-12-myristate-13-acetate Ptdlns Phosphoinositide PtdIns(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate PtdIns(4)P Phosphatidylinositol 4-phosphate PtdIns(4,5)P2 Phosphatidylinositol 4,5-bisphosphate PLC Phospholipase C PP2A Protein phosphatase 2A PRK PKC-related kinase PRR Pattern recognition receptors PTEN Phosphatase and tensin homolog deleted on chromosome ten PX Phox homology R A C Related to A and C kinases RAPTOR Regulatory associated protein of mTOR Ras-GRP Ras guanyl nucleotide-releasing proteins RBC Red blood cell RhoGDI Rho GDP dissociation inhibitor RICTOR Rapamycin-insensitive companion of mTOR RIP Receptor interacting protein RISC RNA-induced silencing complex R N A i R N A interference ROS Reactive oxygen species S6K1 p70 S6 kinase 1 SARA Smad anchor for receptor activation SH2 Src-homology 2 SH3 Src-homology 3 SHIP SH2 domain-containing 5'-inositol phosphatase Xlll shRNA. Short hairpin RNA ' siRNA Small interference RNA TAB TAK-binding protein T A K TGF -P activated kinase. T A N K TRAF-familiy-member-associated N F K B activator kinase TAPP Tandem PH domain-containing protein TBK-1 TANK-binding kinase 1 TGF-p Transforming growth factor-P Th T helper • TIR Toll/IL-1 receptor TIRAP TIR-domain-containing adaptor protein TLR Toll-like receptors TNF-a Tumor necrosis factor-a TRAF TNF receptor associated factor T R A M TRIF-related adaptor molecule TRIF Toll-IL-1 receptor domain-containing adaptor inducing IFN-P TSC Tuberous sclerosis complex UTR Untranslated region VAMP3 Vesicle-associated membrane protein 3 VDR Vitamin D receptor Z Zymosan X I V A C K N O W L E D G M E N T S I offer my sincere gratitude to my supervisor, Dr. Neil Reiner, for the opportunity and guidance in my studies. His enthusiasm and drive for scientific inquiry will have a lasting impression on me. I thank all members of the Reiner lab, Drs. Devki Nandan, Martin Lopez, Alireza Moeenrezakhanlou, and Sanaa Noubir, who have offered me scientific help, support, and friendship throughout the years. I will miss our always interesting and stimulating conversations. I also want to thank Drs. Zakaria Hmama, Alice Mui, Khalid Sendide, and William Nauseef for their technical help and collaborations on various projects. And to Linda Ip and Jane Lee, I thank them for streamlining the administrative side of things for me. To the members of my supervisory committee, Drs. Roger Brownsey, Anthony Chow, Gerald Krystal, and Susan Porter, I thank them for their input and encouragement. I like to thank my parents, who have supported me throughout my years of education. To my sister, Iris, I thank her for instilling in my childhood an appreciation of the value of scientific discoveries. And lastly to C.A.H. , I thank her for the encouragement through these years. xv C H A P T E R I: I N T R O D U C T I O N 1.1 M o n o n u c l e a r p h a g o c y t e s 1.1.1 M a c r o p h a g e o r i g i n , m o r p h o l o g y , a n d h e t e r o g e n e i t y C e l l s o f t h e m o n o n u c l e a r p h a g o c y t e l i n e a g e are h e t e r o g e n e o u s i n t h e i r p h e n o t y p e d u e t o t h e i r w i d e s p r e a d t i s s u e d i s t r i b u t i o n , m o r p h o l o g y , a n d f u n c t i o n . A m o n g t h e s e c e l l s are i n c l u d e d b l o o d m o n o c y t e s , K u p f f e r c e l l s i n l i v e r , L a n g e r h a n s c e l l s i n t h e s k i n , o s t e o c l a s t s i n b o n e , m i c r o g l i a i n t h e c e n t r a l n e r v o u s s y s t e m , a l v e o l a r m a c r o p h a g e s i n t h e l u n g , a n d t i s s u e m a c r o p h a g e s i n l y m p h n o d e s . T o e m p h a s i z e t h e i r c o m m o n l i n e a g e , t h e t e r m m o n o n u c l e a r p h a g o c y t e s y s t e m ( M P S ) i n c l u d e s p r o g e n i t o r c e l l s i n t h e b o n e m a r r o w , 1 2 b l o o d m o n o c y t e s , a n d r e s i d e n t t i s s u e m a c r o p h a g e s ' (Fig. 1-1). E v e n t h o u g h n o t a l l c e l l s i n t h e M P S a r e t r u l y m o n o n u c l e a r — o s t e o c l a s t s , e p i t h e l i o i d c e l l s , a n d g i a n t c e l l s are a l l m u l t i n u c l e a t e d — t h e t e r m p l a c e s t h e e m p h a s i s o n t h e f a c t t h a t t h e m a j o r i t y o f c e l l d i v i s i o n o c c u r s i n t h e m o n o b l a s t a n d p r o m o n o c y t e s t a g e , w h i l e t i s s u e m a c r o p h a g e s h a v e v e r y l i m i t e d r o l e s i n t h e m a i n t e n a n c e o f r e s i d e n t t i s s u e m a c r o p h a g e p o p u l a t i o n s . M a c r o p h a g e s a r e p h y l o g e n e t i c a l l y p r i m i t i v e , b e i n g f o u n d i n a l l v e r t e b r a t e s , c o n s e r v e d t h r o u g h o u t m e t a z o a , a n d e v e n s o m e p r o t o z o a e x h i b i t f e a t u r e s s i m i l a r t o t h e m a m m a l i a n m a c r o p h a g e . A l t h o u g h c e l l s o f t h e m a c r o p h a g e s y s t e m a r e u s u a l l y t h o u g h t o f as p r i n c i p a l l y i m m u n e c e l l s , i t h a s b e e n p o s t u l a t e d that c e l l s o f t h e M P S o r i g i n a l l y e v o l v e d to m e e t a f u n d a m e n t a l n e e d f o r t h e i n t e g r i t y o f m u l t i c e l l u l a r o r g a n i s m s . I n t h i s m o d e l , m u l t i c e l l u l a r o r g a n i s m s r e q u i r e d c e l l s l i k e m a c r o p h a g e s t o e l i m i n a t e d a m a g e d o r a b n o r m a l c e l l s a n d t h e i r p r o d u c t s , t h u s m a i n t a i n i n g h o m e o s t a s i s a n d s t r u c t u r a l i n t e g r i t y . 1 Host defense functions of these cells evolved as a secondary gain to this requirement for multicellular! ty. Figure 1-1A 5M-CFU Monoblast Promonocyte Monocyte] G M - C S F , M - C S F , IL-3, KIT Lymph node t Myeloid dendritic cells Resident macrophages | Langerhans cells (skin) -*• Kupffer cells (liver) Osteoclasts (bone) Microglias (CNS) Innate activation Humoral activation Classical activation Alternative activation ed macrophages> Deactivation Selectins, Chemokines, Integrins, ICAM-1 1-1B M-CSF Tissue macrophages Fusion Clonal expansion Osteoclast/giant cell Figure 1-1. Schematic diagram showing differentiation, distribution, and activation of macrophages and multinucleated giant cells. A, Tissue macrophages originate from bone marrow hematopoietic stem cells (HSC) that have the capacity for self-renewal. Under the influence of linage-determining cytokines (e.g. GM-CSF, M-CSF) and stromal interactions, HSC give rise to short-term HSC which differentiate through common myeloid progenitors and then granulocyte macrophage progenitors (GM-CFU). Further differentiation and expansion then gives rise to monoblasts, promonocytes, and finally monocytes that leave the bone marrow to enter the blood. In the blood, monocytes are recruited to tissue via a multistep process: margination due to hemodynamic changes, rolling, of monocytes on endothelium occurs through low affinity selectin-ligand interactions, activation of monocytes by chemokines leads to expression of high-affinity integrins resulting in stable adherence to endothelium, and finally transmigration through the vessel wall at inter-endothelial cell junctions. Resident macrophages in different organs adapt to the local microenvironment, and differentiate into sub-lineages. B, Tissue macrophages fuse and become multinucleated, and then differentiate into osteoclasts or giant cells. Tissue macrophages must undergo clonal expansion first in response to M-CSF, Adapted from Ref. 4 ' 5 . 3 Monocytopoiesis begins in the bone marrow in humans, and the most immature cell of the MPS is the monoblast, which like all cells of the hematopoietic system has its ultimate origin from pluripotent hematopoietic stem cells 6 (Fig. 1-1A). Various cytokines (e.g. GM-CSF, M-CSF, and IL-3) have been identified as having a regulatory-role in monocytopoiesis. The presence of these mediators and stromal interactions leads to further differentiation and proliferation into promonocytes and then monocytes. These monocytes leave the bone marrow to enter the bloodstream. Studies in mice have shown that the half-life of monocytes in blood is about 17h under normal circumstances 7 , while o longer times of up to 70h have been reported in human . In adult humans, about 1-6 % of total circulating white blood cells are monocytes, rarely exceeding 10% under normal conditions 6 . Monocytes enter the tissues by first rolling along the endothelium via low-affinity interactions, followed by high-affinity adherence to endothelium, and then diapedesis between endothelial cells (Fig. 1-1A). In the tissues, depending on the microenvironment monocytes differentiate into specific subpopulations with distinct functions, thus generating heterogeneity. This seeding of monocytes to different tissues is thought to be a random process 6 . A particularly interesting ability of macrophages is syncytium formation, which is known to be mediated in part through CD47 and macrophage fusion receptor (MFR) interactions (Fig. 1-1B) 5 . Multinucleation has two main functions. First, the increase in cell size permits the elimination of large targets via extracellular degradation, a process that has been described as an 'extracellular lysosome', analogous to intracellular lysosomes 9 . This can occur when the macrophages are not able to ingest the particle. For example in 4 chronic inflammatory states directed at large foreign bodies, macrophages fuse and differentiate into multinucleated giant cells in an attempt to clear the debris. A second function of multinucleation is that it endows macrophages with enhanced capacity, such as amplified bone resorption by osteoclasts. This contrasts with mononucleated macrophages which cannot resorb bone efficiently 5 . Differentiation into osteoclasts is highly regulated and involves a vast array of genes 1 0 . 1.1.2 Macrophages respond to heterogeneous stimuli through a group of diverse cell surface receptors Macrophages are able respond to diverse stimulus due to the expression of a large number of cell surface receptors 6 . This ability to recognize a wide range of exogenous and endogenous ligands and to respond to them appropriately is fundamental to macrophage function in host defense (both innate and adaptive immunity), homeostasis, inflammation, autoimmunity, and immunopathology 1 1 . Immune recognition receptors are diverse, and in addition to receptors for numerous cytokines, macrophages express receptors for non-opsonic recognition of microbes (CD14/LBP, TLR, CR3), phagocytosis of opsonized particles (FcyR, CR3), adherence (integrins), chemokines (CCR2), and recognition of apoptotic cells (CD36) (Table 1-1); Among innate immune receptors, pattern recognition receptors (PRR) have been studied extensively in recent years. PRR are used by the host to detect microbial infection, and the structures that these receptors recognize are called pathogen-associated microbial patterns (PAMP). Examples of these include bacterial lipopolysaccharide (LPS), lipoteichoic acid, peptidoglycan, lipopeptide, dsRNA, ssRNA, bacterial flagellin, CpG D N A and.others 1 2 . Some investigators have 5 argued that the terms PRR and P A M P are a bit misleading, since there is no absolute requirement for a true molecular pattern per se, such as multivalent crosslinking, or the presence of any repeating units ' 3 . Rather, PAMP may be more aptly characterized as particular molecular sequences found within microbial macromolecules and conserved amongst diverse species which may or may not be pathogenic. Furthermore, some PRR have been shown to bind endogenous ligands like fibrinogen or heat shock proteins 1 4 . However, when looking at the Toll-like receptors (TLR) family as a whole, each particular microbe—based on its array of PAMPs—has the potential to activate several 13 different TLR simultaneously, such that an innate "immune signature" is formed . Such a pattern may allow the host to modulate an appropriate immune response towards a given type or class of microorganism. The discovery of PRR began with research on the Drosophila Toll protein. Toll was known to be involved in establishing dorsal-ventral polarity during embryogenesis, and was later found to be important in host defense against fungal infections in adult flies [reviewed in Ref. 1 5 ] . The cytoplasmic domain of the Drosophila Toll is very similar to the mammalian IL-1 receptor, and these domains are referred to as Toll/IL-1 receptor (TIR) domain. It was actually this similarity that prompted investigation of the Toll pathway in regulating immune responses 1 6 . Since the discovery of Drosophila Toll's role in host defense, subsequent research has identified a family of structurally related proteins related to Toll, and these collectively have been referred to as Toll-like receptors (TLR) (Fig. 1-2). Of the thirteen mammalian 6 TLR genes that have been identified, ten are expressed in human . Each of these germline-encoded, non-clonal receptors can recognize a particular set of PAMPs, and thus they represent a primary group of sensors used by the host to detect pathogens. Two key, common structural features of TLRs are an extracellular leucine-rich repeat (LRR) domain and an intracellular TIR domain I 5 . Recent crystal structure analysis of TLR3 revealed that the ligand binding site resides in the C-terminal half of the LRR, while the N-terminal half of L R R is believed to interact with other coreceptor or accessory molecules 1 8 ' 1 9 . Monocytes and macrophages express mRNA for most TLR except TLR3, which is expressed in dendritic cells 2 0 . The major ligands for most of the TLR have been identified. A partial list is shown in Table 1-2. Besides structural specificity achieved from the. existence of diverse TLR, ligand specificity and diversity can be further broadened through heterodimerization between TLR. For example TLR2 can heterodimerize with either TLR1 or TLR6, resulting in different ligand specificity (Fig. 1-2). Some TLR like TLR1, 2, 4, and 5 are expressed on the cell surface, while some are endosomal (TLR3, 8) and can detect ligands inside endosomes 2 1 . Another way diversity can be achieved is through associations with non-TLR receptors, such as the situation in which TLR2 dimerizes with dectin-1, a C-type lectin that enables TLR2/dectin-l to recognize yeast wall particles called zymosans 2 2 ' 2 3 . Activation of TLRs results in overlapping and distinct cellular responses in macrophages, dendritic cells, and B cells. The downstream responses include production of inflammatory mediators, secretion of cytokines and chemokines, upregulation of costimulatory molecules, and transcription of antiviral genes 1 5 . 7 While TLR are clearly a major focus of innate immunity research, it should be noted that other non-TLR PRRs are known to be important in innate recognition. These include f-methionyl-leucyl-phenylalanyl (fMLP) receptor, a G protein coupled receptor (GPCR) on neutrophils involved in chemotaxis. A deficiency of fMLP receptors in mice increases susceptibility to Listeria infections 2 4 . Another important class of sensors is the intracellular pathogen sensors belonging to the nucleotide-binding oligomerization domain (NOD) protein family 2 1 . NOD proteins are structurally similar to the plant R proteins, that are involved in resistance to infection. Missense mutations in the human nod2 gene are associated with the pathogenesis of inflammatory diseases like Crohn's disease and Blau syndrome 2 5 . These highlight how dysregulation of the innate immune recognition system can lead to inflammation, autoimmunity and impaired pathogen clearance. 8 Table 1-1 Receptor family Example Functions Scavenger (collagenous) SR-A Phagocytosis of bacteria and apoptotic cells, endocytosis of modified LDL. adhesion Scavenger (nonco Hag e nous) CD36 Phagocytosis of apoptotic cells, diacyl lipid recognition of bacteria GPl-gncfiored CD 14 iPS-binding protein. TLRfM[?2/MyDS8 interactions, apoptotic cell recognition Integrin CR3(CD11WCD18) CR-mediated phagocytosis, adhesion to endothelium lg Superfamily FcR (ITAMJITIM) Antibody-dBpBndent binding, phagocytosis, killing Seven transmembrane CCR2 C5aR RecBptor forcriBmokine MCP-1 Cbemotaxis, degrsnulation NK-like C-type lectin-like Dectin-1 p-ojucan receptor, fungal particle ingestion C-type lecti n (single CTLD) DC-SIGN Dendritic cell pathogen recognition, ICAM adhesion Multiple CTLD Mannose Receptor Clearance, alternative activation, antigen transport Toll-like receptors TLR2 TLR4 Response to peptidoglycan, zymosan Response to LPS Table 1-1. Overview of macrophage receptors in immune recognition. Adapted from i i 9 Figure 1-2 A Tri-acyl lipopeptide ? ' ; a n C p y ' i r t e L P S Flagellin ? Uropathogenic lipopeptide a bacteria u 1 I dsRNA Imidazoqul nolines ssRNA C p G D N A Hemozoin • I TLR4 TLR5 TLR 10 T L R ^ TLR2TLR6 (can dimerize •I with TLR1and2) TLR1 TLR2 Endosome Leucine-rich repeats TIR domain TLR 9 TLR3 TLR7 TLR8 Figure 1-2. Toll-like receptor family and their principle ligands. T L R 2 can heterodimerize with either TLR1 or T L R 6 , resulting in different ligand specificity. T L R 2 can also dimerize with non-TLR receptors, such as the lectin, dectin-1, to recognize zymosan. Murine TLR11 recognizes uropathogenic bacterial products. TLR11 is a pseudogene in humans 1 7 . T L R 3 , T L R 7 , T L R 8 and T L R 9 are localized to endosomal membranes, and recognize nucleic acids. T L R 9 can also recognize non-nucleic acids such as hemozoin, a breakdown product of hemoglobin produced by Plasmodia spp. Only representative ligands are shown. TLR10 is an orphan receptor that is able to 26 21 homodimerize and also heterodimerize with TLR1 and T L R 2 . Adapted from Ref. . 10 Table 1-2 Receptor Ligand TLR1 Triacyl lipopeptides TLR2 Peptidoglycans, Lipoteichoic acid Lipoarabinomannan Atypical LPS TLR3 dsRNA TLR4 LPS Taxol HSP 60, 70 Fibrinogen TLR5 Flagellin TLR6 Zymosan Diacyl lipopeptides TLR7 Imidazoquinoline ssRNA Loxorebine TLR8 Imidazoquinoline ssRNA TLR9 C p G D N A TLR10 unknown TLR11 unknown, from uropathogenic bacteria Table 1-2. Toll-like receptors and their ligands. Adapted from Ref. 11 1.1.3 Activation of macrophages Macrophages can acquire new or enhanced functional capacities when "activated" in response to various agonists 2 7 . This may lead to enhanced cytokine and chemokine expression, phagocytic capacity, cellular metabolism, responses to chemoattractant signals as well as acquisition of antimicrobial or antitumor functions, and modulation of the capacity to process and present antigens 4 ' 2 7 . In contrast to "activation", the term "priming" refers to prior exposure of a cell to a soluble agonist that alters its response to a subsequent stimulus 2 8 ' 2 9 . Priming stimuli do not result in an activated phenotype per se, but rather sensitize the cell such that it can then respond to subthreshold stimuli. The 30 classical macrophage priming stimulus is low dose IFN-y , but exposures to other mediators such as TNF-a or proteinases can also prime ' ' . Generally, it is not the chemical nature of the stimulus that determines i f it activates or primes, but rather it is the concentration of the stimulus along with other factors such as co-secreted molecules or adhesive interactions with neighboring cells 3 2 . Priming is not restricted to cells of the MPS, as it has also been observed in neutrophils, basophils, lymphocytes, and eosinophils 32 In general macrophage activation may be rapid and take effect within seconds to minutes, such as with the generation of reactive oxygen species and release of arachidonic acid metabolites 2 7 ' 3 3 . Activation can also lead to adaptive changes that require gene 27 expression and this process occurs over periods of hours to days . Macrophage activation has been classified into five types: innate activation, humoral activation, classical activation, alternate activation, and deactivation [reviewed in Ref . 4 , 1 1 > 3 0 ] . Innate 12 activation involves stimulation by microbial products like LPS, through Toll-like and other pattern-recognition receptors. This may result in secretion of pro-inflammatory cytokines, reactive oxygen species and nitric oxide followed by a regulated anti-inflammatory response. Humoral activation occurs via Fc receptors or complement receptors, and results in cytolytic activity and cytokine secretion. Classical activation is mediated by initial stimulation with IFN-y followed by a microbial trigger (e.g. LPS), which results in the secretion of pro-inflammatory cytokines, upregulation of M H C class II, and an oxidative burst. Alternate activation involves IL-4 and IL-13, which mediate their actions through the IL-4/IL-13 common receptor a-chain (IL-4Ra) 4 . Alternate activation of macrophages biases towards humoral immunity (Th2) favoring allergic and anti-parasite responses, upregulation of M H C class II molecules and mannose receptor expression and increased endocytic activity. The fifth type of "activation" is actually "deactivation", and can be brought about by deactivating cytokines (e.g. IL-10, TGF-P), phagocytosis of apoptotic cells, or some pathogens. Macrophage deactivation results in secretion of anti-inflammatory cytokines, and downregulation . of M H C class II. expression. Deactivation plays a role in the resolution of inflammation and in some pathological conditions 4 . Functional properties of macrophages Macrophages have diverse roles in both immunity and tissue homeostasis. These functions can be broadly classified into several areas: phagocytosis and destruction of microorganisms, removal of cellular products including dead and dying cells, chemotaxis, antigen processing and presentation, and secretion of multiple effectors and regulators. 13 Phagocytosis is the receptor-mediated uptake of large particles (>0.5 pm in diameter) into cells 3 4 . Cells with phagocytic capabilities are of myeloid lineage and include cells of the MPS and neutrophils. Recognition of particles is mediated by phagocytic receptors expressed on macrophages, and this enables binding to a wide range of particles leading to phagocytosis. Phagocytosis is a cytoskeletal-based process that requires membrane insertion into nascent phagocytic cups, and the engulfment of particles leads to the generation of the phagosome. Phagosomes are dynamic organelles that undergo extensive luminal and membrane modifications via interactions with diverse endocytic vesicles and the endoplasmic reticulum 3 5 , 3 6 . The process is termed phagosome maturation, and culminates in the formation of a phagolysosome, also termed secondary lysosome. The microbicidal capacities of phagolysosomes have been attributed to their low luminal pH and abundance of hydrolytic enzymes and. reactive oxygen intermediates (ROI) [reviewed in Ref. 3 4 ' 3 7 ] . Recent evidence, however, has led to the suggestion that K + influx into the phagosome is a more important contributor to killing through the T O activation of proteolytic enzymes through hypertonicity and elevated pH . In this model, the action of superoxide and other ROI is not proposed to be direct attack on microbial structures, but rather as a trigger that leads to protease degradation. The actual relative contributions of these two disparate models are controversial 3 9 , and are likely to be resolved with further research. Phagosomes also provide a site for antigen processing 'in and presentation, as well as signaling through PRRs . In this manner, phagosomes are organelles that provide a link between innate and adaptive immunity. Numerous important pathogens have evolved to survive within macrophages by either escaping the 14 phagosome (listeria, shigella, rickettsia) or modifying it in such a manner that they are able to survive within it (mycobacteria, salmonella, brucella, leishmania, toxoplasma) 4 0 . How pathogens escape killing by macrophages is an important research focus since further understanding will be useful in developing novel antimicrobial strategies. Macrophages have important roles in the initiation, maintenance, and resolution of inflammation 4 1 ' 4 2 . Injury or microbial invasion stimulates local connective tissue macrophages to secrete cytokines, such as TNF-a and IL-1, which induce rapid changes in endothelial surfaces essential for margination, arrest, and diapedesis of leukocytes 4 3 . The first leukocytes that are recruited from the blood to sites of inflammation are neutrophils, followed by monocytes. After extravasation into tissues, monocytes differentiate into macrophages and migrate towards the site of injury by chemotaxis. Monocytic cells are able to sense and respond to chemotactic gradients produced during inflammation. Through GPCR, monocytes and macrophages can migrate towards the source of chemokine production. Both endogenous and exogenous substances can act as chemoattractants. The bacterial product fMLP (N-formyl-methionyl-leucyl-phenylalanine), as well as endogenous mediators such as C5a, leukotriene B4 (LTB4), and chemokines (IL-8) are some examples. Activation of macrophages results in secretion of cytokines, chemokines, vasodilators, metalloproteinases, and enhanced phagocytosis and microbicidal killing. A l l of these events contribute to the inflammatory response 4 2 . 15 Macrophages are also involved in embryogenesis, morphogenesis, and tissue repair . Remodeling of tissues and resolution of inflammation or injury requires clearing of cellular debris, and macrophages are involved in these processes partly through phagocytosis. Phagocytosis of apoptotic cells is different from that of microorganisms in 37 * * • that inflammatory responses are not induced with the former . In addition, production of anti-inflammatory cytokines such as TGF-P has been demonstrated to accompany ingestion of apoptotic cells 4 5 . Recognition of apoptotic cells requires some additional receptors that are distinct from pathogen recognition, such as phosphatidylserine •receptors 1 1 . Macrophages also perform functions such as the removal of necrotic tissue, fibrin dissolution, regulation of fibroblast recruitment and growth, connective tissue remodeling 4 , and bone remodeling through resorption by osteoclasts 6 . In this manner, macrophages contribute to tissue homeostasis in both normal and pathogenic states through the engulfment of foreign material and the removal of altered endogenous products. Macrophages and dendritic cells function as antigen-presenting cells, by presenting antigenic peptides to stimulate clonal expansion of B and T cells. In this capacity, cells of the MPS act as bridge to link innate and adaptive immunity responses. Mature DCs are unique in their ability to sensitize naive T cells to peptide antigens, whereas a number of different cell types including macrophages are able to bring about a. secondary response by presenting the corresponding peptide to primed T cells 4 6 . Immature DCs are present in most tissues and are efficient at capturing and processing antigens due to their 16 high endocytic activity. Capturing of antigen signals them to mature and migrate towards lymphoid organs 4 6 . Macrophages have an extraordinary secretory capability. Over one hundred substances have been identified to be secreted by macrophages [reviewed in Ref. 3 ] . These include enzymes (lysozyme, proteases), enzyme inhibitors (a2-macroglobulin, a 1-antitrypsin inhibitor), complement proteins (C1-C5), reactive oxygen intermediates (superoxide, H2O2), cytokines (TNF-a, IL-1, IL-6, IL-10, IL-12, M-CSF, chemokines), coagulation factors (tissue factor, factors II, VII, IX, X , XIII), and arachidonic acid intermediates (PGE2, leukotrienes, PAF) to name only a few. This ability to secrete a diverse and large group of mediators enables the macrophage to participate in a large variety of normal cellular processes, as well as pathological ones. 1.2 Phosphatidylinositide 3-kinase 1.2.1 The PI3K family and 3'-phosphoinositides Phosphatidylinositol 3-kinases (PI3K) constitute a family of lipid kinases that phosphorylate the 3'-hydroxyl of the inositol group of D-wryo-phosphatidylinositol or its derivatives (Fig. 1-3A). Phosphatidylinositol (Ptdlns) is an inositol derivative of phosphatidic acid, and Ptdlns is the most abundant inositol lipid in unstimulated mammalian cells 4 7 . The term phosphoinositides (PI) applies to Ptdlns that has been 48 phosphorylated.at one or more position on the inositol ring . The inositol head group has five free hydroxyl groups, but positions 2 and 6 have not been documented to be esterified with phosphate 4 7 . The 3'-PI metabolites produced as a result of PI3K activity 17 are known to be involved in regulating a multitude of cellular events such as mitogenic responses, insulin signaling, differentiation, apoptosis, cytoskeletal organization, membrane traffic along the exocytic and endocytic pathways (reviewed in Ref. 4 7 ' 4 9 - 5 0 ) ; autophagy 5 1 , and others. The ability of PI3K to affect such diverse cellular functions can be partially explained by the existence of multiple isoforms of PI3K, multiple levels of regulation, and likely subcellular localization of PI3K itself, its metabolites, effectors and targets. PI3K family members have been sub classified based upon their structure and substrate specificity in vitro (Fig. 1-4). Class I isoforms exhibit the broadest degree of substrate selectivity in vitro and phosphorylate multiple forms of Ptdlns, including Ptdlns, PtdIns(4)P, PtdIns(4,5)P2 (Fig. 1 - 3 B ) . In vivo, PtdIns(4,5)P2 is reported to be the most likely substrate leading to the formation of PtdIns(3,4,5)P3 5 2 ' 5 3 . Resting mammalian cells contain a significant level of PtdIns(3)P, but very little in the way of other 3'-PIs 4 7 ' 5 4 . Upon stimulation by agonists such as platelet-derived growth factor (PDGF), PtdIns(3,4,5)P3 and PtdIns(3,4,)P2 levels rise rapidly 5 4 , 5 5 . Class I PI3K is further divided into two subclasses, both of which are known to be activated by cell surface receptors. Class IA PI3K are heterodimers consisting of a regulatory subunit (p85a and its splice variants, and p85P or p55y,) and a 110 kDa (pi 10a, p i 10p, or p i 105 isoforms) catalytic subunit (Fig. 1-4) 4 9 ' 5 0 . Both p i 10a and pl lOp are widely expressed in various tissues, whereas pi 108 is more restricted to leukocytes 5 6 . Class I PI3K catalytic subunits have four characteristic domains or homology regions (HR) 1 to 4, which correspond to the 18 kinase domain, heiical domain, C2 domain, and the Ras binding domain, respectively (Fig. 1-4) 4 1 . Class IA PI3K contains different regulatory subunits encoded by three distinct genes (PIK3R1, PIK3R2, and PIK3R3) which encode proteins that share similar structures (Fig. 1-4) 5 0 . The PIK3R1 gene product gives rise to at least 5 different splice forms (only 3 are shown in Fig. 1-4)57'58. Full length p85a and p85p have N-terminal Src-homology 3 (SH3) domains, and Rac-binding domains (also called breakpoint cluster region homology (BH) domain) flanked by two proline rich regions. The Rac-binding domain is homologous to GTPase accelerating factors (GAP) for Rho family small G proteins, however it lacks GAP activity 5 0 . At the C-termini, the two Src-homology 2 (SH2) domains flank the inter-SH2 (iSH2) region, which contains the binding site for the p i 10 catalytic subunit 4 1 . p85 subunits function as both adaptors and regulators through their multiple domains (Fig. 1-5A) 5 9 . Both p85a and p85p exhibit a wide tissue distribution 5 7 ' 6 0 , although splice variants of p85a are predominantly expressed in skeletal muscles and brain 51. PIK3R3 encodes p55y, which differs from p85a and p85P in that it lacks the N-terminal SH3 and the Rac-binding domain, and has very low expression in peripheral blood leukocytes 6 1 . PI3KR3 can generate two forms of the p55y, p55PIK and p50PIK, through alternative initiation of translation 6 2 . Overall, each distinct regulatory subunit (p85a, p85p, or p55y) is not known to selectively associate with a particular p i 10 catalytic isoform 6 0 . 19 Class IB PI3K is composed of a pllOy catalytic subunit that associates with a plOl adapter encoded by the PIK3R5 gene (Fig. 1-4). The overall structure of p i 10y is similar to that of class IA p i 10 isoforms except that instead of the regulatory binding domain at the amino terminus, a pi01 binding domain exists 5 0 . Other differences include the presence of G(3y binding domains in pllOy 6 3 . Association with pi01 is required for activation of pllOy by Py subunits of heterotrimeric G proteins 6 4 , although it has been reported that it can also be directly stimulated by GPy through binding to distinct sites on pllOy (Fig. 1-4) 6 5 , 6 6 . pllOy is expressed mainly in leukocytes, platelets, and cardiomyocytes 6 7 ' 6 8 . 20 Figure 1-3 diacylgiycerol fatty acid tails lie within inner leaflet of lipid Mayer o" phosphodiester link 6' OH f~ inositol head group 5' [ cytosolic It. PtdIns(3 )^P2 PtdIns(3)P -*4»tdIns(3,4)P2 -<-SHIP2 SHIP PtdIns(3»4,5)P3 PI3K (maiiilyy class III) Ptdlns PI4K PI3K (class I . „ . , , ,. j r . -or class lty£'' ™ " 3 K 1 * ™ % * ' PTEN PtdIns(4)P *• Ptdlns(4,5)P2 PIP5K Figure 1-3. Structure of phosphoinositides and pathways of 3-phosphoinositide synthesis and degradation. A, Structure of phosphatidylinositol. The phosphatidic acid component consists of a glycerol backbone with esterified fatty acids at position 1 and 2, and phosphoric acid at position 3. Arrow indicates the site of PI3K action, and scissor indicates the cleavage site by PLC. B, Pathways of D-3'-phosphoinositides and select Ptdlns kinases and Ptdlns phosphatases. Adapted from Ref. 47>48>50. 21 Figure 1-4 C l a s s IA Regu la tory Isoforms Pro. rich BH (rac binding) p85a p55a All 3 isoforms from the same gene y PIK3R1 rich v Pro. rich i Pro i rich Pro . rich C l a s s IA Catalyt ic Isoforms HR4 Pro. rich —>l r i S H 2 V HR2 Helical domain I C2 HR1 p85p PSK3R2 p55y PIK3R3 p110a P/K3CA p110p PIK3CB P1105 PIK3CD C l a s s IB Catalyt ic and Regu la to ry Subuni t r'6^r\ p110y PIK3CG bindinax Pro. Pro. Pro. rich rich rich_ jpJIOybinQinQJ ; ' ' ^ / " ^ ^ / J M J I M i t e i i i y f l i t e f l C l a s s II Catalyt ic Subun i t p101 P/K3R5 C l a s s III Catalyt ic and Regu la tory Subuni t N-terminal myristoylation Helical domain | Drotein kinase" Heat WD-40 domain I [SRSfteJ L domain C2a PIK3C2A C2p PIK3C2B C2y PIK3C2G hVps34 P/K3C3 p150 P/K3R4 Figure 1-4. Mammalian PI3K catalytic and regulatory subunits. The diagrams are schematic and not drawn to scale. Further splice forms with insertions into the inter-SH2 22 region of p85a and p55a are not shown. Also riot shown are the two different forms of p55y subunits 6 2 . Adapted from Ref . 5 0 ' 6 0 , 6 3 . Class II PDKs contain two C2 domains and are not known to be associated with a regulatory subunit 5 0. The C2 domain was initially thought to provide membrane binding and C a 2 + regulation 6 9 , although subsequent analysis revealed that C2 domains in class II PI3K lack the critical aspartate residues that form the calcium binding pocket 7 0. In vitro, Class II PI3K phosphorylates Ptdlns and PtdIns(4)P 6 9 . They have also been shown to phosphorylate PtdIns(4,5)P2 at low levels when lipid substrates were presented together with phosphatidylserine acting as a carrier 6 9 . Three isoforms (C2a, C2p, and C2y) of mammalian class II PI3K have been characterized ' ' . Both the C2oc and C2p isoforms are expressed ubiquitously, while the C2y is predominantly expressed in the liver 7 2 . In contrast to class I PDKs, which are mainly cytosolic, class II PI3K enzymes 48 are predominantly associated with membrane fractions of cells . C2a and C2p differ in divalent cation dependence in that C2a has a preference for Mg over M n 2 + and C a 2 + and their sensitivity to PI3K inhibitors wortmannin (isolated from Penicillium wortmanni) and the structurally unrelated LY294002 7 3 ' 7 4 . Like the rest of the PI3K family members, C2p is sensitive to wortmannin and LY294002 whereas C2a is resistant to similar concentrations of either inhibitor 6 9 . Class III PI3K, is the orthologue of yeast PI3K Vps34p (vacuolar protein sorting), the only type of PI3K in yeast, and phosphorylates only Ptdlns 7 5 . In mammalian cells, hVps34 is associated with pi50, a myristylated serine/threonine kinase. The 23 myristoylation of p i 50 likely helps target hVps34 to membranes, as is the case in yeast 4 1. Yeast strains with mutations in the gene for Vps34p are defective in directing soluble hydrolases to the vacuole, the yeast equivalent of the lysosome . In mammalian cells, hVps34 also plays a role in vesicle trafficking. Fusion events between early endosomes require the product of hVps34 1 1 : hVps34 is also implicated in regulating degradative pathways in the cell. For example, vesicular trafficking of internalized immune complexes for degradation in the lysosome, macroautophagy, and phagosome maturation have all been shown to depend on class III PI3K 3 5'5 1>7 8. m addition, inhibitory antibodies to hVps34 disrupt post-endocytic trafficking of PDGF receptor 1 9 . Comparisons between major model organisms suggest that class I and II PI3K are present mainly in animals, and the number of isoforms increases with the complexity of the organism (Table 1-3). The yeast genome encodes only class III, and class II PI3K is not found in plants or yeasts, or in the unicellular eukaryotic social amoebae Dictyostelium discoideum 4 7 . Invertebrates generally encode a single isoform of class IA, II, and III, usually with no class IB PI3K. Caenorhabditis elegans and Drosophila melanogaster each have a single regulatory subunit of class I PI3K which share characteristics with the shorter mammalian p50a and p55oc subunits. It has been suggested that the longer molecules p85ct and p85p may have evolved to provide mammals with distinct functions 8 0 . Together, these findings suggest that duplication and diversification has occurred in mammalian evolution with respect to PI3Ks. It has been hypothesized that PI3K diversity in vertebrates was driven by the complex regulatory circuits in the adaptive immune system 8 1 . 24 Table 1-3 Class I Class II Class III Catalytic Adaptor Catalytic Adaptor Mammals p110a, [3, 5 p110y p85a,p85p, p55y p101 C2ct, p, y Vps34p p150 Drosophila melanogaster Dp110 p60 PI3K_68D PI3K_59F Predicted gene CG9746 Caenorhabditis elegans AGE-1 AAP-1 F39B1.1 CEvps34 Predicted gene CAA94175 Dictyostelium discoideum DdPIK.1,2,3 1 DdPIK5 1 Saccharomyces cerevisiae Vps34p Vps15p Plants Vps34p 1 Table 1-3. PI3K family in eukaryotes. *No ortholog found as yet. Adapted form ref 25 1.2.2 Activation and regulation of PI3K Through their regulatory subunits Class IA catalytic isoforms of PI3K are recruited to and activated by cell surface receptors with intrinsic protein tyrosine kinase activity (e.g. growth factor receptors) or receptors coupled to non-receptor tyrosine kinases such as src-family kinases (e.g. B-cell receptor, T-cell receptor, Fc receptors) or Janus kinases (e.g. IL-6 receptors) 4 1 . However, the p i 10 catalytic subunits can also be directly regulated by other mediators. For example, GTP-bound Ras can further augment class IA PI3K activation through direct binding to p i 10 8 2~ 8 4 . The class IA PI3K pllOp isoform has also been shown to be activated downstream of G protein-coupled receptor (GPCR), such as chemokine receptors 8 5 ' 8 6 , and be activated directly by GPy subunits of heterotrimeric G proteins in vitro 6 6 , 8 7 , 8 8 . Class IB PI3K pl lOy/plOl can be activated by f\A f\f\ RR Gpy subunits following stimulation of GPCRs such as fMLP receptor ' '. . ThepllOy catalytic subunit can also be directly regulated by Ras, as is the case for class IA p i 10 isoforms 8 9 ' 9 0 . Comparisons of the kinetic properties between pi 10a and pl lOp revealed interesting differences 9 1 . In terms of lipid kinase activity, p i 10a has a higher Vmax and 25-fold higher Km for Ptdlns than p i 10(3. When PtdIns(4,5)P2 was used as a substrate, similar results were obtained. In thymocyte lysates, p.l 108 lipid kinase activity was intermediate between pi 10a and p i 10p when PtdIns(4,5)P2 was used as a substrate . These findings suggest that kinetic differences, in addition to differential tissue distribution, provide a mechanism to explain differential activity of class IA PI3K isoforms. 26 The diversity of protein interaction domains on the regulatory subunits likely contributes to the ability of multiple signaling proteins to activate class IA PI3K (Fig. 1-5A). Through their SH2-containing regulatory subunits, class IA PI3Ks bind to phosphorylated tyrosine residues that are generated by activated tyrosine kinases in receptors and various receptor-associated adaptor proteins. The class IA regulatory subunits prefer to bind to proteins containing the consensus phosphotyrosine motif pYxxM 9 3 . Phosphotyrosine binding promotes translocation of the cytosolic PI3Ks to the plasma membrane, where they encounter their lipid substrates and Ras. Non-phosphotyrosine-based recruitment mechanisms may also contribute to PI3K activation 9 4 ' 9 5 . Some membrane receptors, such as CD2 and IL-2 receptor, show constitutive association with class IA PI3Ks 9 6 ' 9 7 . A l l mammalian cell types investigated express at least one class IA PI3K isoform, and stimulation of diverse receptor that induce tyrosine kinase activity also leads to class IA PI3K activation 4 7 ' 4 9 ' 9 8 ' 9 9 . Under resting conditions, the regulatory subunits of class IA PI3K have been shown to inhibit the catalytic activity of p i 10 subunits, as well as to protect them from thermal inactivation in vitro, 1 0 ° . Dimerization with p85 increases the half-life of p i 10, compared to monomeric p i 10 1 0 ° . Tonic inhibition of p i 10 by p85 is removed by binding of phosphotyrosine-containing peptides to SH2 domains of p85 1 0 ° . Specifically, the iSH2 domain of p85 is sufficient to bind p l l O a but inhibition of p i 10a requires the presence of both the N-terminal SH2 domain linked to the iSH2 domain 1 0 1 . Binding of phosphopeptides to the N-terminal SH2 results in increased kinase activity. Thus the N -terminal SH2 domain mediates both inhibition of p i 10a and disinhibition by 27 phosphopeptides. In contrast, phosphopeptide binding to the C-terminal SH2 relieves tonic inhibition only in the context of an intact full length p85 1 0 1 . It has also been shown that regulation of class IA PI3K can be mediated by phosphorylation of the Tyr residue within the C-terminal SH2 domain of p85 by Src family tyrosine kinases (Lck and Abl) 1 0 2 Phosphorylat ion of Tyr 6 8 8 leads to increased • class IA PI3K activity 1 0 2 . The N -terminal SH2 domain is able to bind to phospho-Tyr6 8 8in the C-terminal SH2, thus an intramolecular interaction has been proposed . . Furthermore, the C-terminal region of p85 has been demonstrated to block Ras-induced class IA p i 10 activation . An emerging model (Fig. 1-5B) is that phosphotyrosyl-proteins recruit PI3K through interactions with the two SH2 domains of the regulatory subunits, and binding to the N -terminal SH2 domain results in conformational changes that releases inhibition of p i 10 by the regulatory subunit 1 0 1 . Binding of phosphotyrosyl-proteins to the C-terminal SH2 also allows p i 10 to be activated by Ras 8 3 . The phosphorylation of Tyr 6 8 8 in the C-terminal SH2 results in the formation of an intramolecular association, also promoting activation of PI3K. This intramolecular interaction may also promote amplification of the PI3K signal by facilitating the removal of phosphorylated PI3K and freeing the 102 receptor for subsequent association with a new PI3K heterodimer . Since SH2 domain-containing phosphatase 1 (SHP-1) has been shown to dephosphorylate Tyr , the newly detached, phosphorylated PI3K may then be dephosphorylated by SHP-1 (Fig. 1-5B) and return to basal state. This would allow PI3K to be recycled for recruitment to a 102 phosphorylated receptor . 28 In addition to the direct inhibitory effect by the regulatory subunit on p i 10 subunits, free monomeric p85 has also been reported to mediate a signaling function, independent of its regulation on p i 10 subunits 1 0 4 . Kahn and coworkers demonstrated that p85 is more abundant than p i 10 subunits, and a least 30% of p85 exists as a monomer in murine embryonic fibroblasts 1 0 5 . This results in competition between the p85 monomer and the PI3K heterodimer in mediating signaling downstream of insulin-like growth factor-1 (IGF-1) 1 0 5 . Studies in p85 knockout (KO) cells also revealed that activation of Jun N -terminal kinase (INK) by insulin is independent of the associated p i 10 catalytic activity. Instead, this ability to activate JNK is dependent on the N-terminal regions of the p85a or p85p, thus indicating the existence of a p85-dependent but PI3 kinase-independent signaling pathways 1 0 4 . More recently, the molecular balance or ratio, between the regulatory and catalytic subunits of PI3K has been shown to regulate insulin sensitivity in mice 1 0 6 . PI3K activity is required for insulin-induced glucose uptake into muscle and fat cells and inhibition by insulin of glucose production by the liver 1 0 7 ' 1 0 8 . It was a surprising finding, therefore, when mice lacking any isoform of p85 exhibited increased insulin signaling even under conditions where total PI3K levels were reduced 1 0 9 - 1 1 3 . These findings raised the possibility that p85 subunits mediate a negative role in insulin signaling. Cantley et al examined mice that were heterozygous knock outs for either p i 10a, p l lOp or both to examine the effects of reduced p i 10 subunits expression on insulin signaling 1 0 6 . Double heterozygous mutants were used because homozygous knockout for either p i 10a or p i 10P resulted in embryonic death 1 1 4 ; 1 1 5 . They discovered that double heterozygous loss 29 of PI3K catalytic subunits p l lOa and pi 10(3 resulted in mice with mild glucose intolerance and mild hyperinsulinemia in the fasting state, suggesting slightly impaired insulin signaling 1 0 6 . This correlated with a 50% reduction in class IA PI3K protein levels in the cells. Thus, deletion of p85 versus pi 10 subunits can have opposing effects on insulin sensitivity and their ratios have a critical role in determining the set point for in vivo insulin sensitivity (Fig. 1-5C). These findings highlight the importance of not considering p85 knockout to be models of class IA PI3K deficiency. In insulin signaling, both mammalian target of rapamycin (mTOR) and p70 S 6 k i n a s e 1 (S6K1) have been implicated in the negative regulation of the PI3K-Akt pathway [reviewed in Ref. 1 1 6 ] . mTOR is a highly conserved protein kinase and is crucial in promoting ribosome biogenesis and cell growth " 7 . mTOR activity is regulated by at least three main inputs: nutrients, energy metabolism, and growth factors . S6K1 is a serine/threonine kinase involved in cell growth and proliferation and its activity is regulated by mTOR 1 1 7 . mTOR exists as a complex with G(3L and regulatory associated protein of mTOR (RAPTOR) 1 1 7 (Fig. 1-6). This complex is sensitive to the compound rapamycin, hence the name of mTOR U 6 . S6K1 has been implicated in phosphorylation of a specific serine residue on insulin receptor substrate-1 (IRS-1), which leads to increased degradation IRS-1 and decreased insulin receptor interaction with IRS-1 1 1 6 ' 1 1 8 . S6K1 also has a negative effect on IRS-1 gene transcription 1 1 9 . In this model, insulin or IGF-1 binding to the insulin receptor leads to the recruitment of IRS and activation of class IA PI3K. The PtdIns(3,4,5)P3 generated as a result then recruits and activates PDK-1 and Akt. Akt phosphorylates and inactivates tuberous sclerosis complex 1 and 2 (TSC-30 1/2), a protein complex with a GAP activity that inhibits the mTOR/S6Kl pathway through inhibition of the upstream activator Rheb GTPase (Fig. 1-6) 1 2 ° . By inactivating TSC1/2, inhibition of the mTOR/S6Kl pathway is removed leading to serine phosphorylation of IRS-1 by S6K1. The resulting degradation of IRS-1 leads to reduced recruitment and activation of PI3K. This negative regulation of PI3K activity selectively uncouples the stimulatory effects of insulin, and does not affect PI3K activation by other stimuli such as PDGF 1 1 6 . This example provides a mechanistic and conceptual framework of how class I PI3K can be regulated at the level of activation by a specific negative feedback pathway, while leaving intact the activation of PI3K by other stimuli. Recently, the class III PI3K hVps34 has been demonstrated as a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to the mTOR and S6K1 pathway 121,122 j n t k j s m o d e l 5 hVps34 is also required for insulin stimulation of S6K1 in addition to class I PI3K, but hVps34 itself is not regulated by insulin . Instead, nutrient status such as low amino acid or glucose starvation, and activation of AMP-activated protein kinase (AMPK) inhibits hVps34 (Fig. 1-6). This inhibition prevents hVPS34 from activating mTOR, and hence activation of S6K1 as well. Thus, the two main inputs to S6K1 activation are independently regulated by distinct PI3K classes: class I regulates the growth factor stimulation component, and class III regulates the amino acid and glucose sensing component. This illustrates how different classes of PI3K can cooperatively regulate a common pathway. 31 The catalytic subunits of class I and III PI3K also have in vitro protein-serine kinase functions, in addition to lipid kinase activities 4 8 , 6 ° . The potential roles of these protein kinase activities have not been investigated to the same extent as has the lipid kinase function of PI3K. Since classical PI3K inhibitors wortmannin and LY294002 interfere 123 124 • 1 equally with both the lipid kinase and protein kinase activities ' , it cannot be assumed that PI3K signals solely by generating 3'-phosphoinositides. In p i 108, autophosphorylation of the catalytic subunit on serine residues results in inhibition of the 125 lipid kinase activity, and this activity was also blocked by PI3K inhibitors . Similar 126 autophosphorylation has also been reported for p i 1 Op and pllOy Autophosphorylation of p i 10B resulted in down-regulated PI3K lipid kinase activity. However, no inhibitory effect of pllOy autophosphorylation on its lipid kinase activity was observed . Protein kinase activity has also been shown for p i 10a, but rather than 127 128 autophosphorylation, it phosphorylates p85a at the pllO-binding domain ' . This inhibitory phosphorylation is increased following insulin stimulation and provides a 123 negative feedback mechanism to regulate PI3K activity . . Serine phosphorylation of the p85 subunit caused a 3 to 7 fold decrease in PI3K lipid kinase activity . In terms of protein kinase kinetics, p i 10a has a higher F m a x and Km towards peptide substrates than does pi 10p 9 1 . Taken together, these findings support a role for inter-subunit and intra-catalytic subunit serine phosphorylation in the regulation of the PI3K, and differences amongst various isoforms may contribute to differential regulation (Fig. 1-5A). Although IRS-1 has been reported to be a substrate of PI3K protein kinase activity downstream of insulin 1 2 4 and IFN-a signaling 1 3 0 , further research is required to clarify the functional importance of these modification. A role for protein kinase activity in 32 class IB PI3K has also been described 1 3 1 . Using COS-7 cells expressing lipid kinase inactive p i 10y, M A P kinase activation in response to lysophosphatidic acid was shown to 131 be dependent on pllOy protein kinase activity . The importance and physiological relevance of the protein kinase activities of various PI3R isoforms remains to be clarified further. Class II PI3Ks are activated by numerous cell surface receptors. For example, the CC chemokine receptor-2 (CCR2) and the insulin receptor activate C2a, while epidermal growth factor receptor (EGFR) activates both C2a and C2p 1 3 2 " 1 3 4 . C2a can also be activated by TNF-a and leptin 1 3 5 . Insulin preferentially activates C2a rather than C2p isoform of PI3K 1 3 6 . The precise mechanism of class II PI3K regulation is not well understood. Although there is a Ras binding domain on the N-terminal region (Fig. 1-4), there is no evidence for binding or regulation by Ras 7 1 . However, deletion of the C2 domain of C2p increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain 7 1. Considerably more is known about regulation of class III PI3K. hVps34 is complexed with the protein kinase pi50, and association with pi50 is required for activation of hVps34 1 3 7 . The product of class III PI3K, PtdIns(3)P, is constitutively produced in both yeast and mammalian cells and total levels do not change significantly in response to cell stimulation with PDGF 5 2 " 5 4 . In contrast, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are almost absent in resting cells 5 3 ' 5 4 . Nearly constant levels of PtdIns(3)P suggest that the subcellular localization of PtdIns(3)P production regulates various hVPS34 dependent 33 cellular processes. In addition to regulation by amino acid or A M P K in the context of mTOR/S6Kl signaling, as discussed previously, class III hVPS34 is also regulated by the Rab family of GTPases in several vesicle trafficking processes. On early endosomes, hVps34 interacts with Rab5 GTPase to generate PtdIns(3)P, and the resultant lipid then recruits tethering and docking machinery to facilitate the fusion of early endosomes 1 3 8 " 1 4 0 . In late endosomal trafficking, the late endosomal protein Rab7 GTPase is known to influence interactions between early and late endosomes 1 4 1 . It has been demonstrated that the inactive GDP-bound Rab7 colocalizes and interacts with hVps34 via pi50, while the active GTP-bound Rab7 dissociates from hVps34/pl50 1 4 2 . Dissociated hVps34/pl50 then becomes active on late endosomes, and mediates late endosomal transport. Therefore membrane recruitment of pl50/hVps34 to the late endosome does not appear to be the point of regulation, rather it is the dissociation from Rab7 that determines the activity of hVps34 1 4 2 . Since a larger fraction of hVps34 is complexed with Rab7 than Rab5, it has been proposed that Rab7 is the critical regulator in the localized control of hVps34 function l 4 2 . 34 Figure 1-5A Rab4, 5, 6 Rac1 cdc42 Membranes Other binding partners: SHIP, Cbl. ezrin Src family Inhibition of kinase activity Ptdlns(4,5)P2 Auto and trans protein phosphorylation 35 Figure 1-5C Wildtype p85 deficient p110 deficient p85 overexpression S tab le , posi t ive mediator L e s s stable negat ive mediator I Pi3 * @ @ & © @ @ 9 § pe? § • • • • • Effect on Insulin r esponse N o r m a l Hypersensi t iv i ty R e s i s t a n c e R e s i s t a n c e Figure 1-5. Regulation of class IA PI3K. A, The intermolecular and intramolecular regulation, catalytic activities, and substrate interactions of class IA PI3K heterodimers. The protein kinase activity of pi 10 subunits can lead to the downregulation of the lipid kinase activity of the complex. This occurs either by phosphorylation of the inter-SH2 region of p85 (in the case of pi 10a) or autophosphorylation (in the case of pllOp and pi 108). Class IA regulatory subunits stabilize the pi 10 subunit and inhibit their catalytic activities. This inhibition can be relieved by engagement of the SH2 domains of the regulatory subunits with phosphotyrosine residues in receptors or adaptors. Some of the known proteins that are known to bind p85 through interactions via various domains of p85 are shown. Adapted from Ref. 6 0 . B, Model of the effect of phosphorylation of / O O Tyr in p85 on PI3K activity, and formation of an intramolecular interaction between the two SH2 domains. In the basal state, Tyr 6 8 is not phosphorylated. Once recruited by phosphotyrosine residues on receptors or adaptors, conformational changes lead to an 36 active PI3K. Binding of the C-terminal SH2 also relieves the inhibition on the Ras binding domain, such that PI3K can be further activated by Ras, which is membrane 688 bound (not shown). Src-family kinases such as Lck can also phosphorylate Tyr and induce an intramolecular complex between the N - S H 2 and C-SH2 domains. This form is active, and becomes detached form the receptors such that other PI3K heterodimers can become recruited, thus amplifying the PI3K signaling. SHP-1 can dephosphorylate T y r 6 8 8 , restoring P I3K to its basal state. Adapted from Ref. 8 3 > 1 0 1 ' 1 0 2 . Q Model of the molecular balance of p i 10 and p85 in mediating insulin signaling. Insulin sensitivity is regulated by the ratio of the positive acting heterodimer p 8 5 / p l l 0 , and the negative regulator free p85. A given ratio between p85/pl 10 and free p85 defines the normal state of insulin sensitivity in wild-type animals. In p85a + /~ or p85f3_/~ mice, free p85 is decreased preferentially. This causes the balance between p85/pl 10 and free p85 to shift towards the positive mediator p85 /p l l0 , which causes increased insulin sensitivity in mutant mice. In contrast, i n p i 10a + /" p i 10p+/~ mice the p85/pl 10 pool is preferentially decreased over free p85. In this case the balance is shifted towards the negative regulator free p85, resulting in decreased insulin sensitivity. Alternatively, overexpression of p85 leads to increase in free p85 and therefore also shifts the balance towards negative regulation, resulting in decreased insulin sensitivity. Adapted from Ref. 1 0 6 . 37 Figure 1-6 lnsulin/IGF-1 Figure 1-6. Control of insulin and PI3K signaling by mTOR and S6K1. Insulin or IGF-1 stimulates the activation of class IA P I3K/PDK1 pathway via IRS-1, leading to the activation of Akt by a Th r 3 0 8 phosphorylation. Ful l activation of Ak t requires Ser 4 7 3 phosphorylation as wel l , mediated by the m T O R / G p L / R I C T O R complex. Activated Akt then phosphorylates and inactivates TSC1/2, leading to the activation of Rheb GTPase, m T O R / G p L / R A P T O R , and then S6K. Th r 3 0 8 and Ser 4 7 3 can be dephosphorylated by protein phosphatase 2 A (PP2A) and P H domain leucine-rich repeat protein phosphatase (PHLPP) , respectively 1 4 3 ' 1 4 4 . S6K negatively regulates the ability of IRS-1 to transduce 38 the insulin or IGF-1 signal. Class III PI3K hVps34 can integrate nutrient input, such amino acids, to mTOR7S6K. Adapted from Ref. U 6 ' 1 2 1 ' 1 2 2 ' 1 4 4 " 1 4 7 . 39 1.2.3 PI3K Effectors ' The products of class I PI3K are lipid second messengers that control a wide range of cellular responses via diverse downstream effectors. Levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 rise sharply in cells after cell stimulation, and they interact with an array of protein effectors that contain pleckstrin homology (PH) domains 4 1 . PH domains are modular segments of approximately 100 amino acids found in many signaling proteins, and a subgroup of PH domains show specificity for Ptdlns(3,4)1*2, PtdIns(3,4,5)P3, or both 1 4 8 - 1 4 9 . PH domains that selectively bind PtdIns(3)P have also been identified 1 5 ° . The empirical finding that some PH domains interact specifically in vitro with PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3 correlates with in vivo data'defining the same PH domain-containing proteins as PI3K effectors 1 5 1 ' 1 5 2 . PH domain-containing effectors include serine kinases (e.g. 3'-phosphoinositide-dependent kinase-1 [PDK1] and protein kinase B [PKB/Akt]), Tec family tyrosine kinases (Bruton tyrosine kinase [Btk] and inducible T-cell kinase [Itk]), phospholipases (PLCy2), adaptor proteins (Gab 1/2 [Grb2-associated binder]), guanine nucleotide exchange factors (GEF) for Rho and A R F family GTPases, and GTPase-activating proteins (GAP) such as centaurins and GAP l m ( F i g . 1-7) 4 8 ; 6 3 . Several PH domains exhibit high affinity for distinct 3'-PI lipids. These include the PH domains of Btk and centaurin-1 which recognize PtdIns(3,4,5)P3 with high affinity and specificity, whereas other PH domains-containing proteins (e.g. tandem PH domain-containing protein-1 [TAPP1/TAPP2]) will only interact with PtdIns(3,4)P2 1 4 8 ' 1 4 9 . Collectively, PH domain-containing proteins are able to propagate and drive downstream signaling events ( F i g . 1-7). 40 One of the most thoroughly studied PI3K effectors is the protein Ser/Thr kinase Akt/PKB (also called R A C [related to A and C kinases]), which translocates to the plasma membrane by binding PtdIns(3,4)P2 or PtdIns(3,4,5)P3 1 5 3 . Full Akt activation requires both specific threonine (Thr 3 0 8) phosphorylation by PDK1 and serine phosphorylation (Ser4 7 3) by PDK2. 4 8 . Activated Akt is then able to participate in diverse signaling pathways including those involved in regulation of cell survival, insulin signaling, cell cycle control, activation of endothelial nitric oxide synthase and others 4 8 ' 6 3 . It has been shown that the activity of PDK1 is also specifically controlled through the binding of PtdIns(3,4,5)P3 or PtdIns(3,4)P2 to its PH domain l 5 4 " 1 5 7 . Furthermore, PDK1 has recently been shown to phosphorylate and activate protein kinases C (PKC) ^ and 8 directly 1 5 8>1 5 9. The exact identity of PDK2 is not clear, and at least ten kinases have been proposed to serve this function 1 4 5 . One of the candidates with strong evidence is mTOR when it is complexed with GpL and rapamycin-insensitive companion of mTOR (RICTOR) (Fig. 1-6) 1 4 6 , 1 4 7 . The mTOR/GpL/RICTOR complex is not sensitive to rapamycin like the mTOR/GpL/RAPTOR complex, and can phosphorylate the critical Ser 4 7 3 on Akt, instead of Thr 3 8 9 on S6K1 1 4 7 . Thus through an interaction with either RAPTOR or RICTOR, mTOR switches its specificity towards S6K1 or Akt, respectively. Phox homology (PX) domains are distinct from PH domains, but are also able to bind various PI 5 0 ' 1 6 0 . P X domains are approximately 120 A A in length and were initially identified through a database search for proteins homologous to the C-terminal region of the class II PI3K C2y 1 6 1 . Examples of P X domain containing proteins with 3'-PI specific 41 binding include the p40 p h o x subunit of N A D P H oxidase and Vam7 which selectively bind PtdIns(3)P, and the p47 p h o x subunit of N A D P H oxidase that selectively binds PtdIns(3,4)P2 I 6 0 ' 1 6 2 . The P X domain containing class II PI3K binds to PtdIns(4,5)P2, but not its 3'-PI product 1 6 2. F Y V E domains selectively bind PtdIns(3)P, and are named after the first four proteins found to contain this motif (Fablp, YOTB, Vaclp, and Early endosomal antigen-1) . The majority of F Y V E domain-containing proteins identified to date are' involved in membrane trafficking (e.g. EEA1 mediates early endosome fusion) (Fig. 1-6) 4 7 . Some F Y V E domain-containing proteins are involved in signaling, such as S A R A (Smad anchor for receptor activation), which can recruit smad transcription factors to the TGFp receptor 1 6 3 . 42 Figure 1-7 5-phosphatases Ptdlns(3,4,5)P3 > Ptdlns (3,4)P2 i r r 4-phosphatases D A G , Ca2+ Protein Apoptosis synthesis regulation Cell cycle Cytoskeletal regulation, c h a n g e s Docking of vesicle coat proteins = PH domain containing proteins Ptdlns(3)P EA1. AR Membrane Signaling trafficking FYVE domain containing proteins Figure 1-7. P I 3 K signal transduction pathways showing interaction of various 3'-PI with PH or F Y V E domain-containing proteins. The exact type of 3'-PI preferred by the P H domain-containing protein is not shown. Examples of G E F s for Rac GTPases include Vav and T i a m l , and A R F GTPases include A R N O , cytohesin-1, and G R P 1 . G A P proteins include G A P l m and G A P 1 I P 4 B P , which are G A P s for Ras GTPase. Abbreviations: A R N O ( A R F nucleotide binding site opener), GRP1 (general receptor for phosphoinositides), Hrs (hepatocyte growth factor regulated tyrosine kinase substrate), FGD1 (faciogenital dysplasia). Adapted from Ref. 48,153,164 43 1.2.4 Negative regulation of PI3K products: Phosphatases The products of class I PI3K are metabolized by two important phosphatases. The tumor suppressor phosphatase and tensin homolog deleted on. chromosome ten (PTEN) and SH2-containing inositol-5'-phosphatase-l (SHIP-1) are important in regulating the levels of PtdIns(3,4,5)P3 in immune cells 8 0 , PTEN removes the 3-phosphate of PtdIns(3,4,5)P3 to generate PtdIns(4,5)P2, while SHIP-1 removes the 5-phosphate to generate PtdIns(3,4)P2 (Fig. 1 - 3 B ) . Knockouts of either of these phosphatases have been created in mice. Mice deficient in SHIP-1 have shorter life spans, and mortality was associated with extensive consolidation of the lungs resulting from infiltration by myeloid cells 1 6 5 . Recent reports of SHIP-1 mutations in acute human leukemias suggests that SHIP has a potential to act as a tumor suppressor by negatively regulating the PI3K/Akt pathway 1 6 6 . SHIP-2 is an isoform that differs from SHIP-1 in its tissue distribution. .SHIP-1 is predominantly expressed in hematopoietic cells, while SHIP-2 is broadly expressed and participates in growth factor stimulated signaling downstream of receptor tyrosine kinases 1 6 7 ' . SHIP-2 has been reported to be important in insulin signaling and diabetes 1 6 8 Knockout of SHIP-2 resulted in increased sensitivity to insulin^, which was characterized by severe neonatal hypoglycemia, dysregulated expression of the genes involved in 1 /TO gluconeogenesis, and perinatal death . However, SHIP-2 has also been shown to play a role in myeloid cells 1 6 9 ' . SHIP-2 is expressed in human alveolar macrophages, and is inducible by LPS in human peripheral blood monocytes - 6 9 . Studies in transfected cell lines suggest that SHIP-2 down regulates NFicB-dependent gene expression and Akt activation in response to Fey receptor Ha (FcyRIIa) clustering 1 6 9 ' . 44 While PTEN _ /" was found to be embryonically lethal 1 7 0 , PTEN + / " mice are viable but have a propensity to form tumors, indicating a balance between PI3K and PTEN is crucial for regulating cell growth in vivo 1 7 1 . Cells and animals deficient in either PTEN or SHIP have revealed important roles for PI3K in a variety of functions. These models are limited, however, in that they cannot be used to directly address which class I isoform is responsible for a particular phenotype since the products of all 4 isoforms of class I PI3K are subject to regulation by PTEN and SHIP. Other 3'-phosphatases include the F Y V E domain containing myotubularin-like phosphatase 1 7 2 , and the yeast Saclp phosphatase l 7 3 . Both of these have been reported to be specific for PtdIns(3)P [reviewed in Ref. 4 7 ] . The precise roles of these phosphatases in regulating PI3K pathways remain to be defined. 1.2.5 Multiple isoforms of PI3K confer diversity in regulation of cellular processes A considerable amount of research has led to the conclusion that flexibility and diversity of cellular control is differentially mediated by distinct PI3K isoforms 4 7 ' 5 0 . Gene targeting of various PI3K isoforms in mice has identified non-overlapping functions 60,63,67,8i,i67,i74 f o u r c j a s s j pj3j^ catalytic subunits and the regulatory subunits have been genetically manipulated in mice (Table 1-4). Overall, an interesting disparity was observed with regards to the viability of the resulting embryos. Knockouts of either p i 10a or p i 10p resulted in embryonic death 1 1 4> 1 1 5 ; while that of p i 108 of p i lOy did not 9 2 , 1 7 5 . These distinctions were likely due to the ubiquitous expression, of p i 10a and cn pi 1 Op, while p i 108 and pllOy are more restricted to hematopoietic cells . Instead of 45 gene knockouts, mutant p i 108 mice have also been created and are useful alternatives . Thus far, however, no phenotypes in monocytic cells from either knockout or mutant p i 108 animals have been reported 9 2 ' 1 7 6 ' 1 7 7 . Macrophages from class IB PI3K pllOy knockout mice, on the other hand, displayed impaired chemotaxis but normal phagocytosis 1 7 5 ' 1 7 8 ' 1 7 9. Knocking out the regulatory subunit p85 has not turned out to be particularly informative in developing an understanding the roles of distinct class IA p i 10 isoforms. For example, in cells from p85a or p85p knockout mice, expression of p i 10 isoforms a, P, and 8 were 180 183 either reduced or normal depending on the cells studied " . Furthermore, although deficient in p85, these mice did not appear to be bona fide PI3K knockouts since rather than showing loss-of-function, PI3K signaling in some pathways was apparently elevated 110,112,113 p Q r e x a m p i e ; p85a knockout mice were paradoxically found to be hypoglycemic and exhibited increased insulin sensitivity " 3 . These results were surprising since class IA PI3K is known to be essential for mediating the effects of 1 OA insulin on glucose homeostasis . In p85p knockout mice, similar hypoinsulinemia, hypoglycemia, and improved insulin sensitivity were also observed . Moreover, insulin-induced activation of PKB/Akt, a downstream effector of PI3K, was upregulated in p85p knockout mice when compared to wild-type counterparts. In the case of p85a knockout mice, it was shown that alternative splice forms of p85a (p55a and p50a), were expressed at higher levels than in wild type controls and this may have accounted for increased insulin-induced generation of PtdIns(3,4,5)P3 1 1 3 . However, 46 mice that were deleted for p85a and all of its splice variants (pan-85a) still displayed enhanced insulin action similar to mice in which only p85a alone had been deleted (Table 1-4) 1 1 °. The paradoxical phenotype of increased insulin sensitivity and hypoglycemia in p85 KO may be explained by the complex interaction between p85 regulatory subunits and pi 10 catalytic subunits 8 3 ' 1 0 5 ' 1 6 7 . F o r example, since p85 normally inhibits the kinase activity of its associated pi 10 subunit in the basal state, removing p85 could result in hyperactive pi 10 subunits 1 1 2 ' 1 7 4 . Furthermore, p85 has been shown to interfere with Ras activation of pi 10 8 3 . Once again, removal of the influence of p85 has the potential to make pi 10 monomers more sensitive to activation by Ras 1 7 4 . Taken together, these findings indicate that studies targeting the class I regulatory subunits have substantial limitations when the aim is to investigate the specific contributions of pi 10 catalytic isoforms to cellular control 47 Subur i t d is rupted KO Viabili ty, Immunological phenotypes Non-immune phenotypes Alterat ion i n PI3K subunit express ion • References p85o&, p56~«, p50a (pan-p85ct) Homozygous: Perinatal death Defects in B cell proliferation development T cell proliferation and development not affected rVfest cells: FccRI degranulation not affected, 4 SCF or IL-3 induced proliferation Hypoglycemia, hypoinsulinemia Tp85p. 4 p 1 1 0 « , 4 -p l lOp. 4p1105 111.182.183 Heterozygous: Viable Hot reported . . Increased glucose tolerance and insulin sensrtivfcy Tp85p. p 110«. and p11 Dp not affected 112 p55oi, p50oc (still express p85<0 Viable Hot reported Increased insulin sensitivity Not reported 110, p85«, (exon 1 A still express p50<t. p55<t) Viable - Impaired B cell development and Kid-like phenotype Increased insulin sensitivity, hypoglycemia t p55cr. and pSOot in muscle and fat cells, p110a. and p110p not affected in insulin sensitive cells. B cells: 4p1106\ p55o.and p5D<t unaffected T cells: T p55<t and p5Di Ivtast cells: p56"<tand pSOa. unaffected, 4p110<t Dendritic cells: p50<t and p85p unaffected, 4 p11Dp Platelets: p55cc.and p50ct unaffected, 4p110ct. pHOp and p i 106 " 114,181,184, 186-188 p85p Vfable T cells: T proliferation and survival B cells and mast cells: not affected Increased insulin sensitivity, hypoglycemia. p85ct. p55<t, p50<t. p i 10a. p l l O p . and p1105 not signrficarrtry affected 113 p U O a Bnbryonic lethal (day E9-10) Hot applicable General proliferation defect Heterozygous deletion has no effect. tp85<t 115 p 11Dp Bnbryoriic lethal (day E3-7) Hot applicable ' • c Phenotype description lacking due to very eariy lethality Not reported 116 p i 10a** pi10p*^ Viable Hot reported M i d glucose intolerance and mild hyperinsulinemia 4p85o>p, 4p110<tand p l l O p 107 p1105 ' ' Viable Impaired B cell development, BCR signaling Impaired T c e l l activation and T C R signaling neutrophils: 4-chemotaxis . Knock in with cataryticalry inactive subunit also resulted in: Development of spontaneous inflammatory bowel disease, 4-FCERI degranulation and cytokine production in mast cells Not reported M i d 4p85<t, p55<t, and p50<t in B cells. Knock in: no ateration in other class IAp110. 93.177.189. 190 p l l O y Viable - rVbcrophages: 4chemotaxis Dendritic cells: 4migration, contact and delayed type hypersenslwity neutrophils: T numbers, 4-chemotaxis, oxidative burst Mast cells: 4 F c i R I degranulation 8 cells: not affected T cells: T CD4/CD8thymocyte ratio, 4thymocyte apoptosis, 4-proliferation of T c e l l Increased myocardial contractilry, decreased vascular permeability, resistance to thromboembolism 4 Glc-stimulated insulin secretion T pancreatic p-cell mass, T intraperitoneal insulin tolerance No alteration in class IAp110 69.176.180. 191-195 ' p10t Viable Impaired leukocyte chemotaxis, similar to p110y KO Not reported Not reported , 6 8 1.3 R E S E A R C H OBJECTIVES Mononuclear phagocytes are important regulators and effectors of both the innate and acquired immune responses and they are also prominently involved in inflammation. Consequently, regulation of monocyte function is an intense area of research interest and recent studies have highlighted an important role for phosphoinositides in monocyte cell regulation. The 3'-PI metabolites produced by PI3K family members are known to be involved in regulating a multitude of cellular events such as mitogenic responses, differentiation, apoptosis, cytoskeletal organization, membrane traffic along the exocytic and endocytic pathways 4 7 ' 5 1 , and aspects that are more specific to monocyte function such as phagocytosis, adherence, the phagocyte oxidase, and cytokine secretion 5 1>1 8 5"1 9 2. A central question posed at the outset of this thesis was to determine how specificity is achieved in PI3K signaling for these diverse biological functions in human monocytic cells. An important goal was to determine when these functions are dependent on isoform-mediated specificity or when the isoforms have redundant functions. . It was anticipated that the results of these investigations would help toward understanding the complex and diverse signaling pathways mediated by PI3K in monocytes, and possibly provide new ideas toward novel therapeutic approaches for inflammatory and immunological disorders. The first objective of this thesis was to address the development of a new approach to stable gene silencing in human monocytic cell lines using lentiviral-delivered small interfering. R N A (siRNA). The second objective was to identify the role of the p i 10a isoform in mediating monocyte adherence and phagocytosis. The final objective dealt 49 with examining the role of p i 10a in regulating LPS-induced cytokine production and the monocyte oxidative burst. 1.3.1 Objective 1: To selectively dowriregulate a distinct PI3K isoform using lentiviral-delivered s iRNA. ; Rationale Despite recent progress in understanding the functions of PI3K family members in various cell types, study of distinct functions of individual PI3K isoform in monocytes has been difficult due to their resistance to genetic manipulation. The traditional PI3K inhibitors that are available including LY294002 1 4 , wortmannin 7 3 , and 3-methyladenine (3-MA) 5 1> 1 9 3 5 are not useful in assigning function to specific isoforms because at effective concentrations they inhibit, virtually all classes of the PI3K family except for class II PI3K C2a 6 9 . One way to obviate this problem has been to rescue PI3K inhibitor-induced phenotypes by delivering either class I PI3K or class III products such as PtdIns(3,4,5)P3 and PtdIns(3)P respectively, by lipid carriers into cells 5 1 . However, this approach also cannot provide insight into the roles of distinct "class I PI3K isoforms. Another non-genetic approach to study the function of specific PI3K enzymes has been microinjection of inhibitory antibodies. This has proven to be useful for examining the roles of PI3K isoforms when combined with imaging studies of single cells, such as the murine macrophage cell line J774 1 9 4 '. However, not every type of cell can be subjected to this technique 1 9 5 and due to the limited number of cells that can be studied, biochemical characterization is virtually impossible. In addition, this method is transient 50 in nature, and cannot generate a stable cell population for phenotype analysis for a prolonged period of time. Genetic approaches to assigning function to individual class I PI3K p i 10 isoforms have been limited as well since gene knockouts of p i 10a or p i 10P in mice were found to be embryonically lethal 1 1 4 , 1 1 5 . Consequently, it has not been possible to determine with precision the roles of these isoforms in immune cells. Due to the close and complex relationships between class IA regulatory and catalytic subunits 8 3> 1 0 5 ' 1 6 7 ; an ideal strategy in studying the functions of specific PDKs would be to reduce the expression of individual isoforms without disturbing the molecular balance of the regulatory and catalytic subunits. This has not always been achievable. For example, p i 10a embryonic knockout cells had increased p85a expression 1 1 4 , and the massive accumulation of p85a monomers observed was suggested to exert a dominant negative effect on the remaining class IA p i 10 isoforms by binding non-productively to receptors 1 0 5 ' 1 U . Conversely, in cells from p85a or p85p knockout mice, expression of p i 10 isoforms a, P, and 8 was 180 183 either reduced or normal depending on the cells studied " . A genetic approach that has had success in monocytic cell lines is viral-mediated transduction. Lentiviruses in particular, have been shown to transduce at >90% efficiency 1 9 6 ~ 1 9 8 . R N A interference (RNAi) is a sequence-specific post-transcriptional gene silencing mechanism initiated by the introduction of double-stranded R N A (dsRNA) into target cells 1 9 9 . By using a lentiviral vector, we attempted to generate monocytic cell lines that stably expressed siRNAs targeting, the p i 10a isoform of P D K . This approach .51 allowed us to utilize, an efficient gene delivery method coupled with the ability to stably silence a specific PI3K isoform. 1.3.2 Objective 2: Examining the role of p i 10a isoform in monocyte adherence, FcyR-and CR3-mediated phagocytosis. Rationale l a , 25-dihydroxycholecalciferol (D3) is a biologically active from of vitamin D and plays an important role in numerous cellular and physiological processes such as calcium 200 202 homeostasis and regulates cells of the hematopoietic system [reviewed in Ref. " ]. For example, D 3 induces maturation markers such as CD1 lb and CD 14 in monocytic cell lines such as THP-1, U-937 and HL-60 1 8 7 - 2 0 3 - 2 0 6 . I n previous work from this laboratory, D 3 was observed to activate PI3K, and PI3K activity was shown to be required for the induction of CD1 lb and CD14 expression by D 3 1 8 7. D 3 also induced adherence in cells of the human promonocytic cell line THP-1 2 0 3 ' 2 0 7 ' 2 0 8 ; although it is not known whether this involved PI3K. Bacterial lipopolysaccharide (LPS) is also known to enhance 209 210 188211 adherence of leukocytes in vitro ' and it also activates PI3K in monocytic cells ' . 192 212 We and others have previously shown that LPS induced adherence in THP-1 cells 2 1 4 and that this was PI3K dependent192. However, the roles of individual PI3K isoforms in this phenotype have not yet been defined. Using THP-1 cells deficient in p i 10a, in this thesis we investigated whether p i 10a regulates D 3 or LPS-induced adherence or both, and whether CD1 lb upregulation by D 3 is dependent on p i 10a. 52 Phagocytosis plays a pivotal role in host defense against infection. Several lines of strong evidence have indicated a role for PI3K, particularly class I A , in mediating phagocytosis in macrophages. Pharmacologic inactivation of PI3K using either wortmannin or LY294002, blocked particle internalization during FcyR- or complement receptor 3 (CR3)-mediated phagocytosis 2 1 5 " 2 1 7 . Since wortmannin and LY294002 inhibit all isoforms of PI3Ks with the exception of class II PI3K C2ct, it was not possible based upon these studies to specifically assign this function to one particular enzyme class. Another question is whether regulation of FcyR-mediated phagocytosis by PI3K is receptor-specific, or whether phagocytosis mediated by CR3 is similarly regulated. Several observations favor the involvement of class IA PI3K in regulating FcyR-mediated phagocytosis. Non-phagocytic cells transfected with PI3K p85-FcyRIa chimeric receptor were rendered phagocytic, further supporting a role of class IA PI3K in regulating FcyRI-• 9 1 R mediated phagocytosis . Furthermore, phagocytosis via FcyR was associated with, increased activity of the class IA enzyme and with increased levels of the class I product 9 1 Q 99 1 PtdIns(3,4,5)P3 in nascent phagosomal cups " . More recent evidence supporting a role for pllOp in mediating phagocytosis in murine macrophages was provided using microinjection of inhibitory antibodies 2 2 2 . The specific roles of different class I PI3K isoforms in regulating phagocytosis in human monocytic cells are uncertain. While 217 indirect evidence suggests that PI3K may also regulate CR3-mediated phagocytosis , this remains to be studied directly while at the same time examining the specific isoforms involved. 53 1.3.3 Objective 3: Role of p i 10a isoform in LPS-induced cytokine secretion and in oxidative burst.' Rationale Prior research from this laboratory and others demonstrated that the bacterial lipopolysaccharide (LPS), activated PI3K 1 8 8>2 1 1. Otherwise known as endotoxin, LPS is recognized to be a potent agonist leading to cytokine production by monocytes, including TNF-a, IL-lp, IL-6, IL-10, IL-12, IL-8 and many other chemokines [reviewed in Ref. 2 2 3 ] . Several reports in the literature demonstrated a role for PI3K in regulating LPS-induced cytokine production ' . For example, pre-treatment of human monocytic cells with PI3K inhibitors prior to LPS suggested that PI3K negatively regulates TNF-a production 1 9 1 . Bone marrow derived dendritic cells from p85a knockout mice were found to have enhanced IL-12 secretion in response to LPS, here again suggesting that class IA PI3K 228 limits proinflammatory cytokine production downstream of TLR4 . These findings, however, are based upon global inhibition of nearly all PI3K isoforms and may also be limited by non-specific effects of inhibitors and knockouts. In fact, knowledge about the roles of individual PI3K isoform in regulating LPS-induced cytokine production in human monocytes is not available thus far. Here, we investigated the role of PI3K pi 10a isoform in regulating monocyte cytokine production in response to LPS. One important component of the host innate immune response to infection is the phagocyte oxidative burst. This results in the generation of reactive oxygen intermediates (ROI) and microbicidal activity 2 2 9 . ROI in phagocytes include OCI", -OH, 54 and H2O2, which are derived from the superoxide (CV) generated by phagocyte N A D P H (nicotinamide adenine dinucleotide phosphate, reduced) oxidase . Mutations in the genes encoding the N A D P H oxidase subunits in humans results in chronic granulomatous disease (CGD), which is characterized by the absence or very low levels of superoxide 230 production in phagocytes and susceptibility to recurrent bacterial and fungal infections Pharmacological inhibition or genetic studies have demonstrated that PI3K is required for the activation of the neutrophil N A D P H oxidase when stimulated by fMLP, C5a, IL-8, or opsonized zymosan 1 7 5> 1 7 8. 1 7 9- 2 3 1- 2 3 4 When PI3K pi\0y''' neutrophils were incubated with chemoattractant they did not produce PtdIns(3,4,5)P3, did not activate Akt, and displayed both impaired oxidative burst activity and chemotaxis 1 7 5 . On the other hand, superoxide production in response to the particulate agonist opsonized zymosan was intact in pllOy KO neutrophils. Notably, the opsonized zymosan-induced oxidative burst in pllOy K O neutrophils was sensitive to PI3K inhibitors l 7 5 ' 2 3 1 . This finding, along with increased PI3K activity in phosphotyrosine immunoprecipitates from zymosan stimulated monocytic cells, suggested that a class IA PI3K is required for particle induced oxidative burst responses ' . Using monocytic cells deficient in p i 10a, we examined directly whether the oxidative hurst responses to either opsonized zymosan or the soluble agonist phorbol 12-myristate 13-acetate are dependent upon this class IA PI3K isoform. 55 C H A P T E R II: M A T E R I A L A N D M E T H O D S 2.1 Reagents, chemicals, and cell lines Reagents—RPMI 1640, D M E M , Hanks' balanced salt solution (HBSS), penicillin/streptomycin and 1 M HEPES solution were from Stem Cell Technologies (Vancouver, BC). PMSF, anti-BSA antibody (B-7276), human IgM (1-8260), LPS from Escherichia coli 0111:B4 (L-3012), Polybrene, poly-L-lysine, wortmannin, cytochrome' c from horse heart (C-7752), superoxide dismutase (S-8160), and phorbol 12-myristate 13-acetate (P-1585) were obtained from Sigma-Aldrich (Oakville, ON). LY294002, l a , 25-dihydroxycholecalciferol, and antibodies to human pi 108 were from Calbiochem (San Diego, CA). . Antibody to human p i 10a (clone 19) was from BD Biosciences (Mississauga, ON). Antibodies to human p i 10B and actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p85-N-SH3 antibody was from Upstate Biotechnology (Lake Placid, NY) . RPE conjugated anti-CDllb antibody and RPE conjugated isotype-matched control antibodies were from Caltag Laboratories (San Francisco, CA). Anti-phospho-p38 (Thrl80/Tyrl82), phospho-Erk (Thr202/Tyr204), phospho-JNK (Thrl83/Tyrl85), phospho-NFKB p65 (Ser536), and phospho-Akt (Thr308) antibodies were from Cell Signaling Technology (Beverly, M A ) . Ant i -GAPDH antibody was from Research Diagnostics (Concord, MA) . HRPO conjugated anti-Rabbit, anti-mouse, and anti-goat secondary antibodies were from Cedarlane Laboratories (Hornby, ON). Zymosan A and bovine serum albumin (BSA) were from MP Biomedicals, L L C (Irvine, CA). Protein G Sepharose was from Amersham Biosciences (Piscataway, NJ). 56 Cell lines—-The prbmonocytic cell lines THP-1 and U-937 (ATCC, Rockville, MD) were cultured in RPMI 1640 supplemented with 10% FBS (Life Technologies, Burlington, ON), 2mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 pg/ml). Cultures were maintained without exceeding 0.5 x 106 cells/ml. 293T human embryonic kidney (HEK) cells, were also from A T C C and were cultured in D M E M , supplemented with 10% FBS, 2mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 pg/ml), and 20mM HEPES. 2.2 Monocyte transfection and electroporation with oligonucleotides and siRNA Sense and antisense phosphorothioate-modified oligonucleotides, siRNA duplex— Phosphorothioate-modified oligonucleotides were prepared and incorporated into cells as described previously 2 3 1 . Briefly, phosphorothioate-modified oligonucleotides to the oc-isoformof the p i 10 subunit synthesized by Life Technologies, Inc. Oligonucleotides were phosphorothioate-modified to prevent intracellular degradation and purified by HPLC to remove incomplete, synthesis products. 21-mers were produced to the human a isoform of the p i 10a subunit of PI3K, including the presumed translation initiation site in both sense and antisense directions with the following sequences: sense (5'-ATG CCT C C A A G A C C A T C A TCA-3') and antisense (5'-TGA T G A T G G TCT T G G A G G CAT-3'). THP-1 cells (5 x 106) were resuspended in 500 pi of Opti-MEM I Reduced Serum Medium containing 2.5% Oligofectamine (Life Technologies) . and 5 p M phosphorothioate-modified oligonucleotides and incubated on a rotary shaker for 4 h at 37 °C prior to prior to adherence and differentiation. The 21mer siRNA targeting p i 10a (5'-AAU GCC U C C G U G A G G C U A C A U - 3') with dTdT 3'-overhangs and the 5% 57 fluorescein labeled luciferase GL2 siRNA duplex (Dharmacon, Chicago, IL), were transfected in the same manner as the antisense phosphorothioate-modified oligonucleotides. Electroporation of siRNA—Cultured THP-1 cells were first washed twice with RPMI at room temperature, and each 0.4 cm cuvette (BioRad) contained 5 x 106 cells/400 pi of RPMI. Control siRNA labeled with FITC (Dharmacon, Chicago, IL) was added to a final concentration of 100. nM. A l l electroporations were done in a BioRad electroporation device, set at 975uF, with the voltage varying from 200 to 500V. Cells were chilled on ice for 10 minutes prior to electroporation. After electroporation, cells were transferred to wells containing 5 ml of complete culture medium, and cultured overnight. 2.3 Lentiviral preparation and transduction of monocytic cells Constructs—The U6 promoter vector pSHAG-1 and pSHAG-Ffl were kind gifts from Dr. G.J. Harmon (Cold Spring Harbour Laboratory, Cold Spring Harbour, NY) . pSHAG-1 contains the a#Ll/L2 transposition elements that are compatible with Gateway Cloning Technology (Invitrogen Canada Inc., Burlington, ON). Antisense to.pl 10a mRNA (GenBank NM_006218) was targeted to two nucleotide segments: 5'-ATATACATTCCTGATCTTCCTCGTGCTG-3 ' (nucleotide positions 1171 to 1198, referred to as a3), and 5 ' - C A A G A C C A T C A T C A G G T G A A C T G T G G G G - 3 ' (nucleotide positions 8 to 35, referred to as a l ) . The hairpin containing sequence was created as described 2 3 8 . Oligonucleotides p l l O a l and p l l0a3 listed in Table I were synthesized by Qiagen Inc. (Valencia, CA). A l l of the sequences contained a Hindlll site in the 58 hairpin region, a site that is not present in the native pSHAG-1 vector, and BamHI and BseRl ends to enable directional cloning. The oligonucleotides were annealed and then ligated into pSHAG-1 via the BamEI/BseRl site. DH5a E. coli (Invitrogen) were transformed and clones were screened by Hindlll digestion. pSHAG-Ffl contains a U6 driven sequence that generates an hairpin RNA that targets GL3 firefly luciferase at nucleotide positions 1619 to 1647 (GenBank U47296) 2 3 9 . Construction of the lentiviral transducing plasmid, pHR-CMV-EGFP, packaging vector pCMVAR8.2, and V S V envelope vector pMD.G have been described elsewhere 2 4 0> 2 4 1. Purified pSHAG-1, pSHAG-pl 10al, and pSHAG-pl 10a3 served as entry clones. The lentiviral transducing vector pHR-CMV-EGFP was modified by inserting the Gateway vector conversion cassette (Invitrogen) in the Clal site, that is located downstream of 5'LTR, but upstream of the C M V promoter. The resulting pHR-Gateway served as a destination vector since it contained attRl/2 sites. The various entry clones were transposed to the pHR-Gateway by Gateway L R Clonase Enzyme Mix (Invitrogen). Positive clones were then isolated and the plasmids (pHR-U6, pHR-p l lOa l , pHR-pll0a3) purified. A l l plasmid purifications were carried out using Qiagen Endofree Plasmid kits. 59 Table 2-1 C o n s t r u c t 5 ' -An t i s ense sequence.(28nt) ha r ip in loop (8 nt) Sense sequence (28nt) Terminat ion (6 T ' s ) -3 ' p 'HOo.1 C C C C A C A G T T C A C C T G A T G A T G G T C T T G GAA GCTTGC G A G A C C G T C A T C G G G T G A G C T G T G G G G C A T T T T T T T G A T C A A A A A A A T G C C C C A C A G C T C A C C C G A T G A C G G T C T C G C A 4 G C 7 T C C A A G A C C A T C A T C A G G T G A A C T G T G G G G C G p ! 1 0 a 3 C A G C A C G A G G A A G A T C A G G A A T G T A T A T G ^ W G C T T G A T A T G C A T T C C T G G T C T T C T T C G T G T T G C T C T T T T T T G A T C A A A A A A G A G C A A C A C G A A G A A G A C C A G G A A T G C A T A T CA4GCr7"CAT A T A C A T T C C T G A T C T T C C T C G T G C T G C G Table 2-1. Short Hairpin R N A encoding sequences targeting human p i 10a mRNA. H a i r p i n s e q u e n c e s w i t h t h e Hindlll s i te are u n d e r l i n e d a n d i t a l i c i z e d . F o r e a c h c o n s t r u c t , t h e t w o s t r a n d s o f D N A w e r e a n n e a l e d a n d l i g a t e d i n t o t h e p S H A G - 1 v e c t o r . T h e u n d e r l i n e d s e q u e n c e s at t h e 5' a n d 3' e n d s are f o r d i r e c t i o n a l c l o n i n g i n t o p S H A G - 1 , w h i c h w a s c u t w i t h BseRl a n d BamHI. 6.0 Lentivirus packaging—The packaging cell line 293T H E K (5 x 106) was plated on poly-L-Lysine coated 100 mm tissue culture plates (Corning) and transfected the following day. Ten pg of the transducing vector pHR (pHR-U6, pHR-pllOocl, or pHR-pll0a3), 7.5 pg of the packaging vector pCMVAR8.2, and 2.5 pg of the V S V envelope pMD.G were co-transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. The media were changed the next day, and cells were cultured for another 24 hr. Conditioned media were then collected and cleared of debris by low speed centrifugation (2,500g for 5 min) filtered through a 0.45 pm filter and stored at -70°C. This collection was repeated daily for three more days, and media from the 4 days were pooled and ultracentrifuged at 100,000 x g, at 4°C for 2 hr. Pellets were resuspended in 500 pi of medium (overnight on a nutator at 4°C), and aliquots were stored at -70°C. Viral stocks were assayed for the p24 core antigen using the Vironostika HIV-1 Antigen ELISA kit (bioMerieux, Inc., Durham, NC) according to the manufacturer's instructions. Titration of lentiviral vectors—1 x 105 293T cells were plated in each well of a six-well plate. On the following day, cells from three wells were removed with cell dissociation solution (Sigma), and counted in order to determine the average number of cells at the time of titration. Three dilutions (1/50,000, 1/5,000, and 1/500) of the concentrated viral stocks were used to transduce the cells. Transduction of 293T cells was done in the presence of transduction adjuvant Polybrene (8 pg/mL). The media were changed 24 hr after transduction. Cells were removed 48 hr post-transduction and analyzed by flow cytometry for GFP expression. The calculation used to determine the titer was transduction units/ml = average cell number at the time of transduction x % of GFP positive cells/100 x dilution factor. 61 Transduction of target cells—1 x IO5 THP-1 or U-937 cells were seeded in each well of a 12-well plate in 500 pi of complete media and transduced by lentiviral vectors at a multiplicity of infection of 10:1. Transduction was carried out in the presence of Polybrene (8 pg/mL). Transduced cells were analyzed by flow cytometry after six days. For transduction of 293T H E K cells, 1 x 105 cells were seeded in each well of a 12-well plate and transduced the next day. Cells were verified for p i 10a deficiency and viral transduction by western blot and flow cytometry analysis, respectively, prior to use in each experiment. 2.4 Western blot analysis Cells were washed once with PBS and lysed in boiling lysis buffer (1% SDS, 50 mM Tris pH7.4, 0.15 M NaCl, ImM NaF, 10 m M PMSF, 1 mM'sodium ortho-vanadate, 1 m M EDTA) for 5 min and passed through a 27 gauge needle. Lysates were cleared by centrifugation at 12,000 x g for 1 min and protein concentration was determined using Bio-Rad DC Protein Assay. Equal amounts of protein were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before transfer to nitrocellulose membranes. Membranes were blocked with 5% skim milk or 3% B S A in TBST for 1 hr at room temperature depending on the antibody. Primary and secondary antibodies were used according to manufacturer's instructions, followed by detection with enhanced chemiluminescence (ECL) technique (Amersham Biosciences, Piscataway, NJ). 2.5 Adherence assays Adherence assay were performed as reported previously with some modifications 1 9 2 . Briefly, 96-well flat bottom culture plates were filled with 5 x 104 THP-1 cells in 200 pi culture medium. 62 Cells were either treated or not with 25 uM LY294002 for 30 min at 37° C, followed by LPS (12.5-500 ng/ml), D 3 (100 nM), or P M A (20 ng/mL). A l l treatments were done in triplicate. LPS was opsonized with 50% normal human serum for 30 min at 37°C prior to use. Plates were incubated for 24 hr (for LPS treatment) or 48 hr (D 3 treatment) at 37° C, 5% C 0 2 . Non-adherent cells were removed by three washes with 200 pi of warm culture medium (37°C). The remaining cells were then fixed with 2% paraformaldehyde/PBS, for 15 min at 37° C. Fixed cells were then washed once and stained with 0.05% crystal violet in 20% methanol for 10 min at room temperature. Dye was removed and the wells were rinsed with water three times. The plates were allowed to dry at room temperature. Cell associated dye was eluted in 100 pi per well of 100% methanol and absorbance was measured at 570 nm in a microtiter plate reader (BioRad Laboratories, Hercules, CA). Absorbance of adherent cells was normalized to P M A treated cells. 2.6 Dual luciferase assays Each well of a 12-well culture plate was seeded with 2 x 105 293T H E K cells and transfected the following day with 0.05 pg pGL3-SV40 and 0.02 pg pRL-TK reporter plasmids (Promega Corp., Madison, Wl) using Lipofectamine 2000 (Invitrogen). Cells were lysed 48 hr later and assayed for luciferase and renilla activity using Dual Luciferase Assay reagent (Promega). Activities are reported as ratios of Photinus pyralis GL3 luciferase (Pp-luc) to Renilla reniformis R L luciferase (Rr-luc). 2.7 Flow cytometry analysis 63 For staining of surface receptors, 1 x 106 cells were washed once with binding buffer (HBSS, 1% FCS, and 0.1% NaN 3), and then stained with anti-CD l i b , FcyRI, FcyRII, or FcyRIII-RPE conjugated antibody, or isotype-matched control-RPE conjugated antibody for 20 min at room temperature. Cells were then washed once with binding buffer and resuspended in 1 ml of binding buffer containing 1.85% paraformaldehyde. Ten thousand cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer. Data were acquired using BD CellQuest software and analyzed using Summit V3.1 software (Cytomation Inc., Fort Collins, CO). 2.8 Cytokine Measurement THP-1 cells at 1 x 106 /ml were stimulated by serum opsonized LPS at 100 ng/ml for the indicated times at 37 °C, 5% CO2. Cultures were subsequently centrifuged at 400 xg for 10 min at 4°C and cell free supernatants were collected and stored at -70°C until use. TNF-a, IL-6, IL-12p40 (BD Biosciences Pharmingen), and IL-10 (eBioscience) in culture supernatants were measured using sandwich enzyme-linked immunosorbent assay (ELISA) using paired cytokine-specific monoclonal Abs according to the manufacturer's instructions. 2.9 RT-PCR Total RNA was prepared using RNeasy kit (Qiagen, Valencia, CA), and RT-PCR was performed using the Superscript First-strand Synthesis System for first strand cDNA synthesis (Life Technologies, Grand Island, NY) . Taq D N A polymerase was from Fermentas (Burlington, ON). The primer sequences for the various cytokines are listed in, the Table 2-2. An initial denaturation for 5 min at 94°C was followed by a cycle of 94°C for 1 min, the desired annealing temperature (see Table 2-2) for 50 seconds, and 74°C for 1 min for extension, with the number 64 of cycles depending on the primer used. The end of the cycle was followed by 10 min at 72°C. PCR products were run on 1% agarose gel in T A E buffer, visualized with ethidium bromide. Table 2-2. Primer sequences and conditions for R T - P C R . mRNA Primer sequences (forward/reverse) Number of cycles Annealing Temp TNF-a T C T C G A A C C C C G A G T G A C A / G G C C C G G C G G T T C A 35 55°C IL-6 A T G A A C T C C T T C T C C A C A A G C G C / G A A G A A G C C C T C A G G C T G G A C T G 35 67°C IL-10 A C G G C G C T G T C A T C G A T T / T T G G A G C T T A T T A A A G G C A T T C T T C 37 56°C IL-12p40 G G A T G C C G T T C A C A A G / C C C A T T C G C T C C A A G A 30 60°C Actin T G A C G G G G T C A C C C A C A C T G T G C C C A T C T A / C T A G A A G C A T T G C G G T G G A C G A T G G A G G G 31 55°C 2.10 Phagocytosis assay U-937 cells were incubated in 6-well plates at 1 xl0 6 /ml , and differentiated with P M A (10 ng/mL) for 48h. Fluorescent Nile Red C M L 6 p M polystyrene latex beads (Interfacial Dynamics Corp., Oregon) were opsonized as follows: For IgG opsonization, rinsed beads were incubated with BSA, followed by anti-BSA IgG. For complement opsonization, human serum was first treated with protein G Sepharose for l h at 4°C with rotation and centrifuged prior to use. The beads were coated with human IgM followed by incubation with protein G-treated and centrifuged serum for 30 min at 37°C. Complement coating was verified by anti-iC3b monoclonal antibody (Quidel Corporation, Mountain View, CA). Prior to adding the particles, the medium was replaced with fresh culture medium and cells were chilled at 4°C for 30 min. After a further 30 min at 4°C, particles were added and phagocytosis was allowed to proceed at 37°C, for lh. Plates were then transferred on ice and non-ingested beads were removed by 65 washing three times with ice cold PBS. Cells were then washed once with binding buffer and resuspended in 1 ml of binding buffer containing 1.85% paraformaldehyde. Ten thousand cells were analyzed using a Becton Dickinson FACSCalibur flow cytometer. Data were acquired using BD CellQuest software and analyzed using Summit V3.1. software (Cytomation Inc., Fort Collins, CO). 2.11 Superoxide assay . Superoxide assays were performed as described previously by measuring superoxide dismutase inhibitable reduction of ferricytochrome c ' , but with several modifications. Briefly, 0.5 x TO6 THP-1 cells were differentiated overnight with lOng/ml (1.6 nM) of P M A . Prior to stimulation, all cells were washed with HBSS twice, and rested in RPMI for 4h at 37°C. Zymosan particles were opsonized or not with pooled human serum for 30 min at 37°C prior to use. Cells were either untreated or stimulated with either 1 uM P M A or zymosan (20:1) in assay, buffer (5.5 m M glucose, 79 u M cytochrome c), in the presence or absence of 30 pg/ml SOD. Each sample was mixed well, and incubated at,37°C. Absorbance readings at 550 nm were done at 30 min post-stimulation, on a BioRad SmartSpec. PBS was used as blank control. 2.12 Statistical analysis For comparison of two treatment groups, two-tailed / test was performed. A j^-value <0.05 was considered significant. For three or more treatment groups, one-way A N O V A was performed on each group and followed by, Tukey test for multiple comparisons. A /?-value <0.05 was considered significant. A l l statistics and graphs were performed using GraphPad Prism software, version 3.0 (GraphPad Software, San Diego, CA). 66 CHAPTER III: LENTIVIRAL-MEDIATED DELIVERY OF siRNA INTO HUMAN MONOCYTIC CELL LINES1 3.1 Introduction Cells of the mononuclear phagocyte series respond to a wide range of diverse stimuli and show complex cell regulation. From the perspectives of cell biology, understanding disease causation and developing novel therapeutics, there continues to be a great deal of interest in understanding how the responses of these cells are regulated. However, study of monocyte and macrophage biology through genetic manipulation by non-viral transfection methods has been challenging [reviewed in Ref. 2 4 5 ] . Methods involving cationic lipid and liposome-mediated delivery of D N A or physical methods such as electroporation result in low transfection efficiency in monocytic cells, loss of viability, and the difficulty of obtaining stable transfection 2 4 6 ' 2 4 7 . An approach that has met with greater success in monocytic cell lines is viral-mediated transduction. While not all viruses can transduce monocytic cells efficiently, lentiviruses have been shown tp do so at >90% efficiency 1 9 6 " 1 9 8 . RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing mechanism initiated by the introduction of double-stranded R N A (dsRNA) into target cells ' " . R N A i is a natural regulatory mechanism that occurs in many organisms including plants, Caenorhabditis elegans, Drosophila, and mammalian cells [reviewed in Ref. ' " ] . The R N A i pathway begins by processing dsRNA into short (< 30 bp) dsRNA duplexes called small-interfering RNA (siRNA) by a host RNAse Dicer. The siRNA then becomes incorporated into a multi-component nuclease 'Most of material presented in this chapter is derived from that published in Ref. 67 complex called the RNA-induced silencing complex (RISC). RISC then uses the siRNA sequence as a guide to recognize cognate mRNAs for degradation. Delivery of siRNAs into mammalian cells by transfection of siRNA or D N A vectors expressing short hairpin R N A (shRNA) has been shown to mediate R N A i successfully ' ~ . Transfection of siRNA is transient, lasting only for a week or so 2 5 a l t h o u g h DNA-based vectors may last longer with drug selection 2 4 9 . In contrast, viral vectors have also been used to deliver siRNA successfully, and these methods tend to provide more stable gene silencing O l f i T^ ' l 0 ^ 1 ' . This section describes how human monocytic cell lines can be effectively transduced using a lentiviral vector to stably silence an endogenous lipid kinase. Other methods such as electroporation or cation lipid-mediated delivery of oligonucleotides or siRNA are presented here for comparison. 3.2 Transfection of anti-sense oligonucleotides and siRNAs into monocytic cell lines using cationic lipids and electroporation Although the delivery of the anti-sense oligonucleotides using Oligofectamine was moderately efficient as assessed using FITC-labeled oligonucleotides (Fig. 3-1 A), silencing of PI3K p i 10a isoform was only modest, achieving around 14 to 45% reduction in protein levels on average (Fig. 3-1B). Not surprisingly this did not result in a detectable phenotype in either phagocytosis or adherence (data not shown). We also tried to transfect siRNA duplexes to initiate RNA silencing of PI3K isoforms. This did not silence PI3K to any greater extent than had the anti-sense oligonucleotide approach (data not shown). 68 Electroporation of siRNA into THP-1 cells was also attempted in order to reduce p i 10a protein levels. Using FITC-labeled siRNA, the efficiency of electroporation was evaluated. Despite evaluating various settings known to be optimal for mammalian cells, the best transfection efficiency achieved was 9.3 %, at 400V and 975pF. No silencing of p i 10a protein was observed using Western blot analysis (data not shown). 69 Figure 3-1A Ltpofectamine only FITC-labeled Oligonucleotides Figure 3-1B Fluorescence CO cp Anti-p110a Anti-actin 7 0 Figure 3-1. Transfection of antisense oligonucleotide into THP-1 cells by Oligofectamine. A, Flow cytometry analysis of THP-1 cells transfected with FITC-labeled anti-sense oligonucleotides (shaded histogram) and with Oligofectamine only (clear histogram). 10,000 cells were analyzed, and the FITC fluorescence intensity was measured on the FL1 channel. Approximately 52% of transfected THP-1 cells were FITC positive. B, Western blot analysis of class IA PI3K p i 10a catalytic subunit in THP-1 cells transfected with anti-sense oligonucleotides. Actin was used as protein loading control. . A l l figures shown are representatives of at least three independent experiments. 71 3.3 Lentiviral-mediated delivery of siRNA Construction of VSV-pseudotyped lentiviral vector expressing shRNA—In order to deliver siRNA into monocytic cell lines, we developed a lentiviral-based vector that expresses a short-hairpin RNA (shRNA) (Fig. 3-2A and B). Vesicular stomatitis virus-pseudotyped lentiviral vectors have been shown previously to be able to transduce GFP into monocytic cell lines 1 9 6 . The V S V glycoprotein G is a substitute for the lentiviral gpl20/gp41 as the viral coat protein since it broadens the range of cell types that can be infected by the virus and also helps to stabilize the virion, yielding higher titers of the virus 2 4 0 ' . Furthermore, by stabilizing the virion, V S V G protein also allows the viral particles to be concentrated by ultracentrifugation, thereby providing higher titers for transduction 2 4 0> 2 5 4. The synthesized sense and anti-sense oligonucleotides encoding the shRNA (Table 2-1) were annealed and then ligated into the BamRl/BseRl site of pSHAG-1 downstream of the R N A Pol Ill-specific U6 promoter (not shown) 2 3 9 ' . The U6 promoter and the sequence encoding shRNA were then subcloned into the viral transducing vector (pHR-U6-shRNA) using Gateway cloning. The resulting plasmid was then used for transient transfection of the packaging cell line H E K 293T (Fig. 3-2A). Transfection of separate plasmids encoding viral structural genes (gag-pol gene products and accessory proteins on pCMVAR8.2, and V S V G glycoprotein on pMD.G) and non-structural sequences (packaging sequence 4*, Rev Responsive Element, and LTRs on pHR-U6-shRNA), ensures that progeny virus and the target cell will not contain any genes that encode viral proteins 2 5 5 . Recombinant viruses were released into the medium and collected every 24 hr for four days. Viral particles were concentrated by ultracentrifugation. Verification of viral production was done by performing p24 antigen ELISA on concentrated viral stocks. A l l viral samples gave high p24 levels ( » 1 6 0 pg/ml). Viral stocks were titered on 293T H E K cells 72 8 9 which became GFP positive when transduced. We routinely obtained 1 x 10 - 1 x 10 transducing units/ml. 73 Figure 3-2A - 5 1 T R 3 3' : shRNA C M V - { ' I M E H ^ B } CMV [ g i g ^ k y- C M V — | vsv G'-V p H R - U 6 - s h R N A PCMVAR8.2 pMD.G Transient oo-transfection of HEK 293T packaging cell 1£Z S - U 6 - D S B H E h CMV - r ^ ^ } f ^ r e } -| Transcription of vector RNA CMV —.gag-pd CMV —[vsyo O Viral structural proteins A Q V Packaging of vector RNAs, assembly and budding of progeny virus Harvest conditioned media every 24 hours for 4 days J 74 Figure 3-2B Transduction of target cells DMA transport to nucleus t \ my "«rr m R N A t a n j e t Processing bf Dcet •nd RISC compl«i into host genome ^ // 1 X I I Transcription mRNA " V X > w > r ^ V H»rp*i formation J liilitlilMiiiQ shRNA H M i l l l . 1 . fjj ^ Translation | ^ GFP Figure 3-2. Construction of a lentiviral vector for transduction of shRNA into target cells. A, To produce the recombinant lentiviral vectors, the packaging cell line H E K 293T was co-transfected by the vector plasmid (pHR-U6-shRNA) , helper plasmid (pCMVAR8.2) , and envelope plasmid (pMD.G) . The general strategy in the production of lentiviral vector-delivered s i R N A is to segregate the rrara-acting sequences that encode for viral proteins from the ex-acting sequences (regions recognized by viral proteins) involved in the transfer of vector sequences encoding the s h R N A [reviewed in Ref. 2 5 5 ] . The vector plasmid contained a U6 promoter-driven s h R N A coding sequence, followed by a CMV-dr i ven enhanced green fluorescent protein (EGFP) reporter. The shRNA nucleotide sequence shown is not specific and is only intended to illustrate a generic shRNA. These elements were flanked by long terminal 75 repeats (LTR), and also contained cw-acting sequences that allowed the vector RNA to be packaged and subsequently, to be reverse transcribed and integrated in the target cell. The packaging sequence Q¥) was only present in the vector plasmid, and not in the other two plasmids. Following transfection, the plasmids pCMVAR8.2 and pMD.G were transcribed downstream of C M V promoters, and they provided the viral structural proteins in trans. These included viral integrase, protease, reverse transcriptase, capsid and matrix proteins, and vesicular stomatitis virus G protein. Together, these proteins act to ^ram-complement the vector by assembling the progeny viral particles, which are limited to a single round of infection. Vector proteins were produced as well, so transfected cells were GFP positive. Conditioned medium was then harvested, concentrated by ultracentrifugation, and stored at -70°C. B, Transduction of target cells was done at a MOI of 10:1. Virions attach at the cell surface via V S V G proteins, fuse with the cell membrane, and release the viral core. Reverse transcription and uncoating of the viral core occurs in the cytoplasm. The dsDNA is. then transported into the nucleus where it integrates. randomly into the target cell genome. Following integration, the U6 and C M V promoters transcribe their respective genes and this results in shRNA production and mRNA for the GFP reporter. The shRNA and mRNAs are exported to the cytoplasm. GFP is then translated, and the shRNA is processed by Dicer and then incorporated into the RNA induced silencing complex (RISC) [reviewed in Ref. 2 3 8 ] . RISC then targets and degrades cognate mRNAs. 76 High efficiency transduction of human monocytic cell lines—THP-1 and U-937 cells were either mock-transduced or transduced with HR-U6, HR-pl l O a l , or HR-pl 10a3 viral stocks at a multiplicity of infection at 10:1. The viral vector RNAs, after being reverse transcribed into dsDNA in the cytosol entered the nucleus and randomly integrated into the genome, thus generating stable cell lines (Fig. 3-2B). Transduced cells expressed GFP and shRNA (GFP only . in HR-U6 vector). Six days post-transduction, cells were analyzed for GFP expression using flow cytorhetry (Fig. 3-3A). The lentiviral vectors were able to consistently transduce >90% of the target cells. 77 Figure 3-3 A *T J$ C^* Q* /IS of* i > r 3» Antt-p110a AntJ-pHOp Anti-p1105 Anti-p85 N-SH3 Antl-actin Anti-p110a AntJ-p110p Am>p1106 Anti-p85 N-SH3 Anti-actirt 78 Figure 3-3. Transduction of monocytic cell lines by lentiviral vectors is efficient and generates stable cell lines deficient in pi 10a. A, Flow cytometry analysis of transduced (solid histogram) or mock transduced cells (clear histogram) was performed 6 days after viral transduction. 10,000 cells were analyzed,.and the GFP fluorescence intensity was measured on the FL1 channel. Approximately 97% of the THP-1 and U-937 cells were GFP positive. The mean fluorescence intensity (MFI) for mock-infected cells was 8.5 for THP-1 and 6.6 for U-937. Transduced cells had MFIs of 38.3 for THP-1 and 132.3 for U-937. B and C, Western blot analysis of class IA PI3K p 110 catalytic subunit isoforms (a, P, and 8), and p85 regulatory subunit in THP-1 cells and U-937 cells. Actin was used as protein loading control, 79 3.4 Silencing of PI3K p i 10a isoform in human monocytic cell lines Stable and specific silencing of Class IA PI 3-kinase pi 10a isoform— shRNA specific to PI3K pi 10a mRNA was used to induce R N A silencing by a mechanism involving the RISC (Fig. 3-2B). Transduced cells were expanded and examined by Western blotting. Figure 3-3B and C show that transduction of THP-1 and U-937 with the HR-pl 10oc3 viral vectors resulted in nearly complete elimination of PI3K p i 10a isoform expression. In contrast, transduction of cells with either lentiviral vector HR-pl 10al expressing a second a l shRNA sequence, U6-promoter control virus, or mock transduction did not affect p i 10a protein levels. The effect of HR-p l 10a3 was specific in that levels of other Class U PI3K catalytic subunits p i 10P and p i 108, or the p85a regulatory subunits were not affected. The stability of p i 10a silencing was confirmed by Western blotting of cells that had been in continuous culture for more than six weeks and of cells stored in liquid nitrogen for at least 2 years. 3.5 Transduced H E K 293T cells can be further transfected with reporter plasmids Stable and specific silencing of luciferase activity in HEK 293T cells—To verify that the hairpin construct from HR-pl 10a3 does not nonspecifically interfere with the transcription of reporter plasmids, we transduced H E K 293T cells with either HR-pl 10a3 or HR-Ff l , which produces a shRNA targeting GL3 firefly luciferase. H E K 293T cells were chosen because they are much more receptive to transfection than are monocytic cell lines. Western blot analysis demonstrated that the level of p i 10a in H E K 293T cells transduced with HR-pl 10a3 viruses was markedly reduced similar to what was observed in similarly treated THP-1 and U-937 cells. As expected transduction of cells with HR-Ffl virus had no effect on p i 10a expression (data not shown). HR-pl 10a3 transduced cells or HR-Ffl transduced cells were then co-transfected with 80 firefly luciferase (pGL3-SV40) and Renilla luciferase (pRL-TK) plasmids, the latter serving as an internal control. Forty-eight hours post-transfection, cells were analyzed using dual luciferase assay (Fig. 3-4). Cell transduction with HR-Ffl reduced firefly luciferase expression by more than 80%, while HR-pl 10a3 had no effect when compared to mock transduced cells. These results indicate that in cells expressing shRNA targeting p i 10a, non-specific interference with the function of a reporter plasmid is not a problem. The data also show that lentiviral-mediated RNAi is capable of silencing exogenous genes driven by a strong promoter. 81 Figure 3-4 Silencing of Firefly Luciferase Figure 3-4. Silencing of an exogenous gene in H E K 293T cells expressing shRNA. H E K 293T cells were transduced or not with viruses targeting PI3K p i 10a (HR-p l 10a3) or firefly luciferase GL3 (HR-F f l ) . After seven days, cells were transfected with firefly luciferase p G L 3 -SV40 and renilla luciferase p R L - T K reporter plasmids. Forty-eight hours after transfection, cells were lysed and analyzed for luciferase activity using Promega's Dual Luciferase Assay. The activities are reported as firefly luciferase (Pp-Luc) /Reni l la luciferase (Rr-Luc). H R - F f l transduced cells gave a Pp-Luc/Rr-Luc ratio < 20% of either mock or H R - p l 10a3 transduced cells (p < 0.01, p o s t - A N O V A Tukey test). Cells transduced with s h R N A targeting p i 10a had similar ratios as non-transduced cells (p > 0.05, p o s t - A N O V A Tukey test). One-way A N O V A for all three cells linesp = 0.0027. Error bars indicate standard deviation, a = 3. 82 3.6 Discussion A major obstacle in studying monocyte cell biology has been the resistance of these cells to genetic manipulation, particularly when using non-viral methods. In the studies described in this thesis, we report a strategy for stable gene silencing in monocytic cells. Using a V S V -pseudotyped lentiviral vector, monocytic cell lines that stably expressed shRNAs targeting an endogenous gene were generated resulting in silencing of the p i 10a isoform of PI3K. Historically, lentiviral vectors have been shown to be superior to non-viral methods such as cationic lipid-mediated delivery of D N A vectors to monocytic cells because of their much higher transduction efficiency and longer period of transgene expression. Although the mechanism underlying the resistance of monocytic cells to D N A transfection is not known precisely, it has been proposed that much of the exogenous D N A enters the cell via endocytosis resulting in degradation of the D N A by abundant lysosomal nucleases 2 4 5> 2 5 6. VSV-pseudotyped lentiviral vectors obviate this problem, since the viral core containing the genetic elements of interest is delivered directly into the cytosol after the viral envelope fuses with the plasma membrane of the j cc target cell . Another physical approach for transfection that has not been very successful in monocytic cells is electroporation. Poor success here has been related to low viability of cells after electroporation, typically below 10-22% survival 2 4 6> 2 4 7. Amongst the various viral-based approaches, lentiviral-based vectors seem to be the most promising for transduction of monocytic cells. Onco-retroviruses are similar to lentiviruses, but the latter have a more complex genome, and consequently more complex replication cycle n c c [reviewed in Ref. ]. One advantage of lentiviral vectors over onco-retroviral vectors lies in their ability to transduce both proliferating and non-proliferating cells, such as liver, muscle, 83 retina, and neurons " . This has been attributed to the presence of nuclear localization signal sequences present in lentiviral gene products 2 6 0 which are absent from onco-retroviral vectors. Interestingly, onco-retroviruses transduce monocytic cell lines THP-1, U-937, and HL-60 at 1 Q*7 1 QS lower efficiencies (1-31%) ' , even though these are proliferating cells. Furthermore, compared to onco-retrpviruses, lentiviral vectors are also much less susceptible to transcriptional silencing of the viral transgene, an event that may result from methylation of foreign D N A in the vicinity of the promoter, as well as by integration of the viral elements into condensed chromatin regions 2 4 5> 2 6 1. Taken together, all of the above differences make lentiviruses potentially superior vectors for the delivery of siRNAs into monocytic cells. Other viruses such as adenoviruses and adeno-associated viruses have also been used to transduce monocytic cells. Adenoviruses do not integrate into the host genome and as a result are not useful for long term expression of the exogenous sequences 2 4 5 . Adeno-associated virus (AAV) in contrast, do integrate into the host genome, and have been used successfully in transducing primary human monocytes and dendritic cells 2 6 2> 2 6 3 ? although their efficacy in transducing human monocytic cell lines has been low (<1%)264. By combining the ability of lentiviral vectors to stably transduce monocytic cell lines at a. high efficiency and the potential for siRNA to mediate R N A interference, we.have shown that stable gene silencing in human monocytic cell lines is achievable (Fig. 3-3). Transduced cells can be propagated under normal conditions without drug selection. The silenced phenotype appears to be stable during 6-8 weeks of continuous culture, and transduced cells may be used after long periods of storage in liquid nitrogen. , 84 In the study of PI3K function, gene-targeting studies in mice have revealed important roles for specific PI3K isoforms in immunity, metabolism and cardiac function 6 0 . However, a particular problem with targeting individual PI3K isoforms has been that silencing of a regulatory or catalytic subunit often results in altered expression of the non-targeted isoforms (Table 1-4) 6 0 . Altered subunit expression often leads to changes in signaling 6 0> 1 6 7 ) and thus makes interpretation of the observed phenotypes complicated. In this context, lentiviral-mediated RNA silencing of p i 10 isoforms appears to be a superior approach, since we were able to specifically reduce pi 10a expression while not affecting levels of either other class IA p i 10 isoforms or p85 (Fig. 3-3B and C). However, it can be argued that since p85 normally heterodimerizes with p i 10, the lack of reduction in p85 in p i 10a deficient cells may suggest that p85 is in relative excess to the total p i 10 in these cells. We cannot exclude this possibility at the present time. Nevertheless, when taken together the results shown indicate that lentiviral-delivered siRNA is an efficient method for specific gene silencing in monocytic cells. Furthermore, by virtue of the specificity offered by R N A i , studies of individual isoforms from protein families can be done with relative ease. In contrast to lentiviral vector HR-pl 10a3, transduction of the HR-pl 10a 1 vector did not reduce cellular levels of p i 10a (Fig. 3-3B and C). While HR-pl 10al was originally designed as a candidate shRNA to mediate R N A i , the sequence did not bring about the desired result. Nevertheless, these cells transduced with HR-pl 10al served as useful controls for non-specific effects of transduction and shRNA expression. There are several possible explanations for why this candidate siRNA might not have been effective. It has been suggested that target mRNA regions where hydrogen bonds form in secondary and tertiary structures can impede silencing 85 265,266 Another possible explanation for the lack of effect of the HR-pl 10a 1 vector may be that this construct targeted a region close to the A U G start codon. It has been suggested that these regions may be richer in sequences that bind regulatory proteins and this may limit the ability of the RISC complex to access the R N A target sequence 1 9 9 ' 2 6 7 . Nevertheless, this is not an absolute restriction since it has been shown that targeting sequences close to the start codon may successfully induce R N A silencing The experiments reported above demonstrated the ability of VSV-pseudotyped lentiviral vectors to stably silence the P I3K p i 10a isoform in the monocytic cell lines THP-1 and U-937. The ability of lentiviral vectors to transduce both dividing and non-dividing cells and stable expression of the transgene make this a versatile strategy for gene silencing based on R N A i . Moreover, the finding that lentiviral transduced cells expressing shRNA can be further manipulated by transfection with reporter plasmids, for instance luciferase, significantly expands the utility of this approach. One important application of this technique may be in gene therapy research, such as silencing genes in myeloid leukemia cells or other difficult to transfect cells in order to better understand their biology and to identify potential therapeutic targets. 86 CHAPTER IV: ROLE OF pi 10a PI3K IN REGULATING MONOCYTE ADHERENCE2 AND PHAGOCYTOSIS 4.1 LPS and vitamin D 3 induced adherence Vitamin D 3 (D3) is well known for its role in regulating calcium homeostasis, however, it also 9 0 0 9 0 9 has other important actions in the regulation of hematopoietic cells " . D 3 has been shown to induce differentiation of monocytic cells, and augment maturation markers such as CD l i b and CD 14 in monocytic cell lines such as THP-1, U-937 and HL-60 1 8 7> 2 0 3- 2 0 6. Previous work from this laboratory showed that D 3 activated PI3K, and PI3K activity was required for the induction of C D l l b and CD14 expression by D 3 1 8 7 . Adherence of monocytes to endothelial cells is an essential requirement for the localization of these cells to sites of tissue inflammation 1 9 2 . Adherence in cells of the human promonocytic cell line THP-1 can also be induced by D 3 203,207,208^ a i m o u g h i t i s n o t c i e a r whether this also involves PI3K. Bacterial LPS is another agonist known to enhance adherence of leukocytes in vitro 2 0 9 ' 2 1 0 j and it * 188 211 also activates PI3K in monocytic cells ' . Numerous reports have previously shown that LPS induces adherence in THP-1 cells 1 9 2 ' 2 1 2 " 2 1 4 j a n c j that this is a PI3K dependent process 1 9 2 . The roles of individual PI3K isoforms involved in this phenotype have not been defined and therefore we sought to use THP-1 cells deficient in p i 10a isoform to investigate whether this PI3K isoform is required for either LPS-induced or D3-induced adherence. Some material presented in this chapter are derived from that published in Ref. 87 C D l l b is the a chain of the p2 integrin Mac-1 (also called complement receptor 3), and Mac-1 initiates multiple cellular processes including adherence, phagocytosis, degranulation, and migration . We have previously reported that C D l l b induction by D 3 in THP-1 cells was sensitive to inhibition by PI3K inhibitors 1 8 7 . The role of PI3K p i 10a isoform in D 3 induced up-regulation of CD1 lb will also be examined in this chapter. 4.1.1 Monocyte adherence induced by D 3 , but not LPS is dependent on p 110a. We have previously shown that LPS-induced adherence of THP-1 cells is dependent upon PI3K 1 9 2 . However, use of pharmacologic inhibitors did not permit determination of whether this involved class IA PI3K and i f so which p i 10 isoform 1 9 2 . To address this question, we examined LPS-induced adherence in THP-1 cells rendered p i 10a deficient using shRNA (Fig. 4A). Consistent with previous findings that PMA-induced monocyte adherence is resistant to PI3K inhibitors I 9 2 , p i 10a deficient cells showed normal PMA-induced adherence (data not shown). This indicated that differentiation of pi 10a deficient cells appear to be normal with P M A stimulation, compared to control transduced, cells. Using PMA-treated cells as a control, adherence induced by LPS was determined as percentage of PMA-induced adherence by a colorimetric based adherence assay as described in Experimental Procedures. Figure 4-1A shows that while PI3K inhibitor LY294002 significantly reduced LPS-induced adherence in all transduced cells, silencing of p i 10a did not affect LPS-induced adherence, and this was'true over a range of LPS concentrations (12.5-500 ng/ml). LPS was opsonized by human serum prior to use, however the presence of human serum alone did not induce adherence (data not shown). Similar to LPS, LY294002 inhibited D3-induced adherence in all types of transduced cells (Fig. 4-1B). In contrast to LPS, however, monocyte adherence induced by D 3 was found to be 88 attenuated in p i 10a deficient cells. Moreover, this inhibitory effect was comparable to that observed in LY294002-treated cells. 89 ure 4-1 LPS-induced adherence 125i S | 100 0) -o N "5 E X E 75 SO 25 0 Anil 12.S il 25 50 LPS (ng/ml) 500 c m Mock • I H R - U 6 •BHR-pHOc/1 • HR-pf 10«3 5 Mock t LY miOU6 + LY ^ H R - p 1 1 0 a 1 + LY FM3 HR-p110a3 + LY B D 3 - induced adherence i i 1 I A 4. V A A * \ / * v * v # # * # >s ^N 90 Figure 4-1. Monocyte adherence induced by D3, but not LPS is dependent on pllOa. A, LPS-induced adherence. Cells were either treated or not with LY294002 (25 pM) for 30 min. at 37° C, before treatment with human serum opsonized LPS (12.5-500 ng/ml). For each treatment group, incubation with P M A (20 ng/ml) was also done in parallel. After 24 hr, non-adherent cells were rinsed away, and adherent cells stained with crystal violet. Dye was then eluted with 100% methanol, and absorbance measured at 570nm. To control for cell numbers plated per cell type, adherence was expressed as a percentage of PMA-induced adherence. PMA-induced adherence was similar among all cell types (p > 0.05, data not shown). A l l samples treated with LPS alone were not significantly different from each other (one-way A N O V A p = 0.2827). The same applies to the LY294002 plus LPS treated group (p = 0.1962). A l l of the LY294002 plus LPS treated samples were significantly different from LPS treated control samples (post-A N O V A Tukey test,/? < 0.01 for all pairs), and the values observed were approximately 40% of the LPS only group. B, D3-induced adherence. Cells were either treated or not with LY294002 (25 uM) for 30 min at 37° C, before treatment with D 3 (100 nM). Cells were incubated with D 3 for 48 hr before being assayed for adherence. A l l samples treated with LY294002 plus D 3 were not significantly different from each other (one-way A N O V A p = 0.5945), while the D 3 treatment alone samples were significantly different from each other (one-way A N O V A p -0.0006). A l l of the LY294002 plus D 3 treated samples were significantly different from cells treated with D 3 only (post-ANOVA Tukey test,/? < 0.05 for all pairs), with the exception of HR-pl 10ot3 (p>0.05). D3-induced adherence in p l l O a deficient cells is approximately 50% of control, and equivalent to treatment with LY294002. A l l treatments were done in triplicate. Error bars indicate standard deviation, n = 3. 91 4.1.2 CD 1 lb expression in response to vitamin D 3 , Di-induced CDllb expression is attenuated in pllOa deficient cells—We have previously shown that CD1 lb induction by D 3 in THP-1 cells was sensitive to inhibition by PI3K inhibitors 1 8 7 . To determine whether this is p i 1 Oa-dependent, we incubated cells with LY294002 or not, followed by D 3 for 72 hr after which the cells were collected and stained with anti-CD 1 lb RPE-conjugated antibody or isotype matched control RPE-conjugated antibody. After washing and resuspension in binding buffer, cells were.analyzed by flow cytometry. Viable cells were gated, and analyzed for RPE signals. For transduced cells, only GFP positive cells were analyzed. As shown in Figure 4-2, D3-induced CD1 lb expression was reduced significantly in p i 10a deficient cells when compared to control cells (p < 0.05). The results shown are expressed as mean fluorescence intensity (MFI) index which is the ratio of [MFI of D 3 treated samples stained with anti-CD 1 lb antibody - MFI of isotype-matched control antibody)/ (MFI of anti-CD 1 lb antibody stained, untreated samples - MFI isotype-matched control antibody]. Therefore, a MFI index of 1 indicates that there was no induction of CD1 lb by D 3 . To calculate % reduction of D3-induced C D l l b expression in p i 10a deficient cells relative to D3-treated control cells, the following calculation was used: % reduction = [(MFI index of control cells - MFI index of HR-pl 10a3 cells)/ (MFI index of control cells - 1)] x 100. The % reduction of D3-induced CD1 lb expression in p i 10a deficient cells relative to controls cells was approximately 59-63%, similar to that observed in LY294002-treated cells. 92 Figure 4-2 Attenuat ion o f D 3 - t n d u c e d C D 1 1 b e x p r e s s i o n in p110a def ic ient T H P - 1 ce l l s 3.0 -) Figure 4-2. C D l l b induction by D3 is attenuated in p l l O a deficient THP-1 cells. THP-1 cells were either incubated or not with LY294002 (25 uM) for 30 min, followed by 100 nM D 3 for 72 h at 37° C and 5% CO2. Cells were then washed once in binding buffer and stained with anti-CDllb RPE-conjugated antibody or isotype matched RPE-conjugated control antibody, according to the manufacturer's instructions. After washing and resuspension in binding buffer containing 1.85% paraformaldehyde, 10,000 cells were analyzed for RPE fluorescence. For transduced cells, the analysis was restricted to GFP positive cells. Data were collected after compensation of GFP and RPE fluorochromes on channels FL1 and FL2. Mean Fluorescence Intensity (MFI) index is the ratio of (MFI of D 3 treated samples stained with anti-CDllb antibody - MFI of isotype-matched control antibody)/ (MFI of anti-CDllb antibody stained, untreated samples - MFI isotype-matched control antibody). Therefore, a MFI index of 1 indicates that there was no induction of CD1 lb by D 3 , or complete inhibition by LY294002. A l l 93 of the LY294002 plus D 3 treated samples were significantly different from control cells treated with D 3 alone (pos t -ANOVA Tukey test,/? < 0.05 for all pairs), but not from H R - p l 10a3 plus D 3 alone (p>0.05). Error bars indicate standard deviation, n = 3. 94 4.2 Regulation of phagocytosis by p i 10a PI3K 9riQ Phagocytosis is the uptake of large (>0.5 pm) particles by cells . This process is triggered by the recognition of ligands exposed on the particle surface by specific phagocyte cell surface receptors. Following receptor binding and crosslinking, actin polymerizes around the phagocytic cup. Phagocytosis also involves bringing about membrane extension or addition to surround the particle, thereby creating an intracellular, particle-containing phagosome 3 6 . In lower eukaryotes such as the slime mold 97ft Dictyostelium, phagocytosis is a process primarily for the acquisition of food . In mammals, the ability to phagocytose efficiently is limited to professional phagocytes (neutrophils and macrophages), cells which play critical roles in host defense, homeostasis, and tissue remodeling. These cells have numerous types of receptors that can mediate binding to a wide variety of ligands. The most well studied type is the Fey receptor (FcyR). Two main classes of these receptors exist, one group activates effector functions, and the second group inhibits these functions. In humans, the former group includes FcyRI, FcyRIIA, and FcyRIIIA [reviewed in Ref. 2 7 1 ] . These receptors differ somewhat in their structures, but they have in common a cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAM) present either in the cytoplasmic domain of the receptor itself (FcyRIIA), or in an associated y subunits (FcyRI and FcyRIIIA). It has been shown that clustering of the FcyRIIA leads to tyrosine phosphorylation of the I T A M motif, present within its cytoplasmic domain, by Src family tyrosine kinases (e.g. Hck 272 * ' and Lyn) . The phosphorylated I T A M then recruits the non-receptor tyrosine kinase Syk via its two SH2 domains. Studies in Syk-deficient mice have shown that Syk is required for completion of FcyR-mediated phagocytosis 2 7 3 . Tyrosine phosphorylated 95 Syk has been shown to recruit PI3K via the SH2 domain of the p85 subunit . The Grb2-associated binder 2 (Gab2), a scaffolding adaptor containing a PH domain, has been proposed to further amplify the PI3K signal by recruiting more PI3K via Gab2's phosphotyrosine residues 2 7 5 . This provides at least one mechanism to physically link class IA PI3K to downstream signaling during FcyR mediated phagocytosis. Additional evidence linking PI3K to FcyR-mediated phagocytosis is the ability of PI3K inhibitors wortmannin and LY294002 to inhibit uptake of IgG-coated RBC or latex beads by macrophages and neutrophils ' ' ' . Inhibition of PI3K did not affect actin polymerization, suggesting that pseudopod extension for particle engulfment is not just 9 1 f\ an actin driven event . Although class IA PI3K has been implicated in regulating phagocytosis based upon its association with Syk in FcyR mediated uptake , other evidence suggests that additional isoforms are also likely to be involved in regulating 990 FcyRI-mediated phagocytosis . For example, Allen and coworkers showed that following FcyRI cross-linking in human monocytes, the activities of both class IA 990 p85/pl 10 and class IB pllOy/101 PI3K were increased . More recently, using microinjection of inhibitory antibodies revealed that pl lOp isoform was required for 999 phagocytosis of IgG-coated RBC and apoptotic cells in murine macrophages . It is unclear whether this finding also applies to human phagocytes or for CR3-mediated phagocytosis as well. The events downstream of PI3K leading to phagosome formation are not well understood. However, current data suggest that PI3K may play a role in mediating contractile activity and membrane insertion required for phagocytosis. Greenberg and 96 colleagues proposed that myosin X provides a molecular link between PI3K and pseudopod extension during phagocytosis 2 1 1 . Myosin X is an unconventional myosin with three PH domains that accumulates in the phagocytic cup in a PI3K dependent 277 manner and is also required for membrane spreading on IgG opsonized particles .. It has been shown that wortmannin and LY294002 sensitivity in phagocytosis of IgG-9 I f. coated beads is dependent on particle size . Thus, internalization of particles larger than 4pm, but not smaller, was attenuated by PI3K inhibitors and this correlated with a reduction in exocytic plasma membrane insertion 2 1 6 . One hypothesis proposed to explain these findings was that larger particles require more membrane to become enclosed, and a specific PI3K may be involved in regulating the delivery of membranes 216 from vesicles to the plasma membrane during phagocytosis . Consistent with the model of targeted delivery of endomembranes contributing to the elongation of pseudopods is that recycling endocytic vesicles bearing the V A M P 3 (vesicle-associated. membrane protein 3) marker have been shown to be delivered at the site of phagocytosis 978 . Furthermore, the small GTP-binding protein ARF6 (ADP ribosylation factor 6) has been implicated in membrane delivery during phagocytosis and expression of dominant negative ARF6 inhibited the recruitment of V A M P 3 + compartments at the site of phagocytosis 2 7 9 . A possible link between ARF6 and PI3K is that an exchange factor for ARF6, ARNO (ARF nucleotide binding site opener), is known to depend on PI3K for 9R0 activation . A R N O also contains a PH domain, and has been shown to bind PI3K products . Thus, in addition to recruiting myosin X , PI3K products may also act to recruit and activate Pi-binding proteins that can stimulate vesicle trafficking required for 97 pseudopod extension. This would provide a unified and elegant mechanism for PI3K to couple exocytosis of endomembrane with myosin X-mediated pseudopod extension. . Phagocytosis mediated by other receptors, such as complement receptor 3 (CR3), has also been shown to be inhibited by PI3K inhibitors 2 X 1 . The mechanism by which PI3K regulates CR3-mediated phagocytosis is less clear, however, vesicles have been observed • 281 • to accumulate around bound particles during CR3-mediated phagocytosis suggesting the possibility of a similar role in exocytosis of membranes 2 8 2 . It is not known which isoform of PI3K regulates CR3-mediated phagocytosis. 4.2.1 Effect of p i 10a silencing on phagocytosis To investigate whether p i 10a regulates FcyR-, CR3-mediated phagocytosis or both, we first determined if PI3K p i 10a knockdown had any effect on the expression of these phagocytic receptors in our model system. To examine expression of phagocytic receptors FcyRI, FcyRII, FcyRIIIA, and CR3 (CD1 lb), flow cytometry analysis was used. The expression of FcyRI, FcyRIIA, FcyRIIIA, and CD1 lb were approximately 83%, 83%, 6.5%, and 87%, respectively, with control isotype matched antibody having <7% staining (Fig. 4-3A). These findings are consistent with previous reports that—with the exception of FcyRIIIA—these receptors are highly expressed on human monocytic cell lines No significant differences were observed between control cells and p i 10a knockdown cells (/?>0.05). Flow cytometry analysis of latex beads was performed to verify their opsonization with either IgG or.iC3b (Fig. 4-3B and C). 98 P M A differentiated U-937 cells deficient in p i 10a had reduced FcyR- and CR3-mediated phagocytosis compared to P M A differentiated control cells (Fig. 4-3D and E). On average, the reduction in FcyR-mediated phagocytosis was 66.5% (SD ±14.3%) compared to control cells, while CR3-mediated was reduced by 55.9% (±20.9%) compared to control cells. Binding of particles to target cells as assessed by exposing the cells to the particles at 4°C for 1.5 h. No differences between the groups were observed 0^>0.05, data not shown). 99 Figure 4-3 B C IgG opsonized S e r u m o p 8 0 n l z e d Figure 4 -3 . Surface receptor expression, opsonization of latex beads, and phagocytosis of IgG and serum opsonized latex beads by PMA-differentiated U - 9 3 7 cells deficient in P I 3 K p i 10a subunit. A , Receptor expression of FcyRI, FcyRII and FcyRIIIA and C D l l b was not affected by silencing PI3K p i 10a. Isotype control antibodies (ctl Ab) , anti-FcyRI (aCD64), anti-FcyRIIA (aCD32), anti-FcyRIIIA (aCD16), or a n t i - C D l l b were used to assess the surface expression of these receptors. The % 101 expression on the Y-axis is the fraction of cells that express the receptor prior to differentiation. No significant difference was seen between p i 10a deficient cells and shRNA control cells (p>0.05). B and C: 6.6 pm latex beads were opsonized with serum or IgG using methods previously described 284 with minor modifications. B, IgG opsonization was achieved using anti-BSA antibodies against BSA-coated latex beads. Verification by flow cytometry was done by using secondary, anti-IgG antibody conjugated to FITC. Clear histogram represents secondary antibody alone, and solid histogram represents anti-BSA IgG followed by secondary antibody. C, IgM coated latex beads were opsonized with complement using fresh serum and this was confirmed with anti-iC3b antibody and FITC-conjugated secondary antibody. Latex beads incubated in the presence of human serum (solid histogram) or incubated in the absence of serum (clear histogram) are shown. D and E: U-937 cells transduced with HR-pl 10al or HR-pl 10a3 were differentiated with P M A for 48 h before exposure to opsonized latex beads for lh. D, Phagocytosis of IgG opsonized latex beads was significantly (p=0.0165, two-tailed paired t test) diminished in p i 10a knockdown cells (transduced by HR-pl 10a3 virus) compared to control cells (HR-pl 10al). E, Phagocytosis of serum opsonized latex beads was significantly (p=0.0225, two-tailed paired t test) diminished in pi 10a knockdown cells (HR-pl 10a3) compared to control, cells (HR-pl 10a 1). Error bars indicate standard deviation, n = 3. 102 4.3 Discussion Vitamin D 3 and LPS are both known to induce adherence in monocytic cells 192,203,207,208,2,2-2,4 ^ t Q p O K 187 , ,88 ,2„ ,285 T h r ( ) u g h ^ ^ Q f ^ assays, we and others have previously shown that both LPS and D 3 activate PI3K in 18*7 188911 98S human monocytes and macrophages ' ' ' . Since in these studies the basic approach used was an anti-p85 antibody to immunoprecipitate the kinase, the results led to the conclusion that class IA PDKs are activated in response to LPS or D 3 , although the involvement of other PI3K family members could not be ruled out. Since pi 10a is a 91 92 286 more robust PI3K in terms of biochemical kinetics than either pi 10p or pi 105 ' ' , we hypothesized that pi 10a might be the dominant class IA PI3K in mediating LPS and D 3 signaling. Based on this assumption, we predicted that by silencing PI3K pi 10a in THP-1 cells, a phenotype of diminished adherence induced by either LPS, D 3 , or both would be observed. As shown in Figures 4 r l A and B, whereas adherence in response to both LPS and D 3 was sensitive to PI3K inhibitor LY294002, LPS-induced adherence was resistant to silencing of pi 10a while D3-induced adherence was not. This was examined over a range of LPS concentrations to control for the possibility that LPS might only utilize pi 10a at lower concentrations. Thus, these findings suggest that differential utilization of pi 10a reflects qualitative differences between LPS and D 3 . These results lead to the somewhat surprising and interesting conclusion that these two agonists use signaling pathways in monocytes that activate distinct isoforms of class IA PDKs at least for some functional responses. It would appear most likely that LPS-induced adherence is mediated by pi 10p or pi 108, since LPS is known to activate class IA PI3K 1 8 8 ' 2 1 1 j and LPS-induced adherence was inhibited by LY294002 ( F i g . 4-1 A)., At this point, we 103 cannot rule out the possibility that LPS activates p i 10a for other signaling pathways not related to adherence. Prior studies from this laboratory showed that in THP-1 cells treated with D 3 , the vitamin D receptor (VDR) associated with the p85 subunit of PI3K in a ligand-dependent manner 1 8 7 . In addition, within 20 minutes of exposure to D 3 , a corresponding rise in PI3K activity was observed when PI3K assays were performed on either.anti-p8 5 or anti-VDR immunoprecipitates. Furthermore, activation of PI3K by D 3 was linked to changes in gene expression after 24 hr 1 8 7 . The findings in the latter and the present reports are consistent with a model in which class IA PI3K is activated through a steroid receptor. This model differs from the conventional paradigm in' which class IA PI3K activation occurs downstream of transmembrane receptors such as growth factor receptors, immunoreceptors [reviewed in Ref. ], and toll-like receptor 2 . However, recent progress in D 3 signaling research has resulted in the detection of a putative membrane bound receptor ( V D R m e m ) , that based upon differences in binding properties, appears to be distinct from the nuclear V D R (VDR n u c ) [reviewed in Ref. ]. For example, selective binding of synthetic D 3 analogs to V D R m e m and not V D R n u c has shown that the former mediates non-genomic rapid signaling effects of D 3 , but not delayed classical genomic responses . Taken together, these findings suggest the interesting possibility that class IA PI3K may be activated by both V D R m e m and V D R n u c , such that PI3K may regulate both rapid, non-genomic signaling as well as at least some delayed genomic effects of D 3 . Clearly in this model there would be ample opportunity for activation of PI3K through V D R m e m to influence cellular responses to D 3 brought about through the classical V D R n u c . 104 Further studies will be required to identify whether V D R m e m complexes with and activates PI3K p85/pl 10a. The P2 integrin receptor CR3 (CDllb/CD18, also known as 0^2), is a marker of monocyte differentiation and can mediate adherence, phagocytosis and leukocyte transmigration [reviewed in Ref. 2 9 0 ] . CD l i b is the a subunit of CR3 and associates non-covalently with its partner CD18. Expression of CR3 is restricted to myeloid cells, and its level of expression depends on the state of differentiation with mature neutrophils 291 293 and macrophages having the highest levels " . THP-1 cells are known to express relatively low levels of CR3 in the basal state 2 8 3 ' 2 9 4 _ 2 9 6 ; a n c j Q 3 is known to augment CR3 expression 187>203>204. We have previously reported that CR3 induction by D 3 in both THP-1 cells and human monocytes is sensitive to PI3K inhibitors 1 8 ? . Using THP-1 cells made deficient in p i 10a by R N A i , we observed a significant reduction (59 to 63%) in D3-induced CD1 lb expression compared to several negative control cell populations (Fig. 4 - 2 ) . Adherence of monocytes to plastic induced by P M A has been shown to be 9Q7 dependent on CR3 . Therefore, the defect in D3-induced adherence we observed in pi 10a deficient THP-1 cells (Fig. 4 - 1 B ) may be partially due to the attenuation of CD1 lb expression. Alternatively, reduced adherence in response to D 3 by p i 10a deficient cells, may be related to defective activation of CR3 since the active conformation of CR3 is 9QR 9QQ required for optimal function ' . In regard to LPS-induced adherence, we have previously reported that another p 2 integrin, LFA-1 (CD1 la/CD18), played an important role in mediating LPS-adherence to ICAM-1 coated plates 1 9 2 . Using neutralizing monoclonal antibodies, we found that blocking C D l l a and CD 18 resulted in 50% and 105 65% decrease in adherence, respectively. When the two antibodies were used in combination, the reduction in LPS-induced adherence was -85%. Since CR3 also shares CD 18 with LFA-1 and can bind to ICAM-1 as well 2 9 0 , this suggests the possibility that CR3 may also mediate LPS-induced adherence to some degree. This hypothesis is strengthened by the fact that LPS also induces C D l l b expression in monocytic cells 295,300 p u r m e r studies are required to investigate the relative roles of various integrins in mediating adherence induced by LPS and D 3 . Phagocytic cells have crucial roles in innate immunity and host defense by virtue of their abilities to recognize, ingest and destroy invading microbes. PI3K has been demonstrated T I C 917 to be an important regulator of both FcyR and CR3-mediated phagocytosis ' . The specific isoform of P O K involved in mediating these events in human monocytic cells is not clear. Using microinjection of inhibitory antibodies, it was shown that p i 10B and to a lesser extent p i 105, but not p i 10a, were required for apoptotic cell and FcyR-mediated phagocytosis of IgG-opsonized RBC in murine macrophages 2 2 2 . These results are at variance with our results in human monocytic cells where we observed that both FcyR-and CR3-mediated phagocytosis were p i 10a PI3K-dependent (Fig. 4-3D and E). There are at least three possible explanations for these discordant findings. First, this inconsistency may reflect species-specific differences in PI3K isoform usage. This possibility is supported by the finding of such species-dependent effects on PI3K isoform usage with effects on phagocyte function in neutrophils 3 1 . Using second generation isoform specific PI3K inhibitors, it was shown that TNF-a primed oxidative burst in 106 human neutrophils in response to fMLP required the sequential activation of pllOy followed by p i 108. In contrast, the oxidative burst in murine neutrophils required only 31 pi 10y and was independent of p 1108 . A second possible explanation may be the differentiation state of the monocytic cells used in our experiments. For example, it has been shown that the P K C isoforms activated by FeyR varies depending on the differentiation state of the cells. In IFN-y primed U-937 monocytes for example, FcyRI engagement led to increased P K C activity and translocation of P K C isoforms 8, e, and while in dibutyryl cAMP differentiated U -937 cells the conventional PKC isoforms (a, P, and y) were recruited 3 0 1 . Therefore it may be possible that the PMA-differentiated U-937 cells in our model may use a different PI3K isoform for phagocytosis than in murine bone-marrow derived or peritoneal macrophages, based upon differences in differentiation state. A third possibility to account for these discordant findings is the difference in the particle system used. It has been shown that mechanical properties of the particle can affect the efficiency of phagocytosis and signaling, independent of size. For example, rigid versus soft polyacrylamide beads showed differential dependence on Racl GTPase 3 0 2 . This variable could be controlled for by comparing experiments using IgG-opsonized RBC as targets. The results shown in Figure 4-3D show that like Fcy-mediated bead ingestion, CR3-mediated phagocytosis also appear to require p i 10a since the uptake of serum-opsonized 107 beads was significantly reduced in p i 10a knockdown cells. These conclusions need to be tempered somewhat by the caveat that in the model systems we used—whereas, events were heavily biased towards either CR3 or FcyR—it was not possible to completely exclude the potential involvement of other receptors. Furthermore, it has been reported that CR3 can form functional associations with other receptors such as CD 14 , FcyRIIIB 3 0 3 , and CD8 7 3 0 4 . A n interesting observation by Grinstein and coworkers was that FcyR crosslinking brought about increased avidity of CR3, and CR3 was mobilized into phagocytic cups during FcyR-mediated phagocytosis 3 0 5 . It has been reported that in 'inf. neutrophils FcyR crosslinking activated CR3 via a PI3K-dependent pathway . Thus, CR3 may participate in FcyR-mediated phagocytosis, even when the particles specifically bind FcyR. This suggests the hypothesis that crosstalk between these two receptors may be altered in p i 10a deficient cells and the possible amplification of a phenotype of defective phagocytosis. Further studies will be required to address this question. Residual phagocytic activity in p i 10a deficient cells (Fig. 4-3D and E) may be due to usage of other PI3K isoforms or may be PI3K-independent. Various studies have suggested that not all phagocytosis requires PI3K. PI3K-independent phagocytosis of a 107 • variety of microbial organisms, including Helicobacter pylori , Yersinia enterocolitica , and Legionella pneumophila has been described. PI3K-independent contractile activity has also been reported as w e l l 3 1 0 . The differentiation state of the cells may also influence whether PI3K regulates phagocytosis 2 6 9 . For example, it has been shown that 7 CO T 1 1 undifferentiated THP-1 cells ingested particles independent of PI3K ' , while FcyR-mediated phagocytosis by retinoic acid/IFN-y differentiated THP-1 cells is inhibitable by 108 PI3K inhibitors . These findings suggest that PI3K may contribute to more efficient phagocytosis in macrophages compared to monocytes 2 6 9 . Basal, PI3K-independent phagocytosis is likely to coexist with the more efficient POK-dependent phagocytosis pathways. In summary, using transduced THP-1 cells deficient in PI3K p i 10a, we found that D 3 -induced, but not LPS-induced adherence is dependent on pl lOa. D3-induced upregulation of GDI lb was also found to be dependent on p i 10a. Therefore our data suggests that p i 10a is required for D3-induced differentiation. Our data also demonstrated a prominent role of p i 10a in mediating FcyR- and CR3-mediated phagocytosis in PMA-differentiated U-937 cells. It should be noted that while P M A -induced differentiation appear to be normal in p i 10a deficient cells, our current data cannot exclude the possibility that other aspects of PMA-induced differentiation besides adherence were affected by p i 10a silencing.. 109 C H A P T E R V: R O L E O F P I 3 K pl lOa IN R E G U L A T I N G C Y T O K I N E PRODUCTION AND T H E O X I D A T I V E BURST 5.1 Toll-like Receptor family Mononuclear phagocytes can detect infection through PRR, which enable these cells to sense diverse microbial ligands (PAMP). These germline encoded, non-cional receptors are critical aspects of host defense against pathogenic microorganisms. The innate immune response is rapid and can modulate the development of the adaptive immune response. A related group of PRR that has sparked tremendous research interest in the last several years is the Toll-like receptor (TLR) family, which was discovered through studies of the Drosophila Toll protein involved in ontogenesis and antimicrobial resistance [reviewed in Ref. 1 5 ] . TLRs are type I transmembrane receptors with leucine-rich repeats in their extracellular domains, while their cytoplasmic domains have homology to the mammalian IL-1 receptor called TIR (Toll/IL-1 receptor) domain 1 6 . TLR stimulation induces a wide variety of genes in macrophages and dendritic cells, including those encoding cytokines, chemokines, proteolytic enzymes, extracellular matrix proteins, and proteins involved in antigen presentation such as M H C class II, and co-stimulatory molecules amongst others [reviewed in Ref. ]. Balancing the induction of pro- and anti-inflammatory responses is an important aspect of TLR signaling, since these are amongst the first receptors stimulated on innate immune cells. For example induction of IL-12 promotes proinflammatory responses, while IL-10 is predominantly anti-inflammatory TLR4 signaling in macrophages 110 The first mammalian TLR discovered was TLR4, and this was accomplished through both a forward genetic approach by positional cloning in a strain of mice that was LPS hyporesponsive and through reverse genetics via TLR4 knockout mice [reviewed in ReL 3 1 2 ] . Active TLR4 is a homodimer, and similar to other TLRs it has a TIR domain in the cytoplasmic region. LPS induced TLR4 signaling is initiated when LPS binds to LPS binding protein (LBP)- in serum, and then to a glycosylphosphatidylinositol (GPI)-anchored CD 14 on the plasma membrane. This complex then presents LPS to the TLR4/MD2 complex on the cell surface. Although currently there is no evidence that LPS binds directly to TLR4 3 1 3 , LPS-induced signaling leading to inflammatory responses clearly involves TLR4. One model proposed is that LPS is recognized by a cluster of receptors associated with lipid rafts 3 1 4 ' . CD 14 and other receptors are constitutively found in lipid rafts, while TLR4 is recruited to lipid rafts upon LPS binding. Following formation of a complex between LPS/LBP/CD14 and TLR4, the latter dimerize and its TIR domains can recruit four TIR domain-containing adaptors through homophilic TIR-TIR domain interactions. These adaptors include MyD88 (myeloid differentiation primary-response protein 88), M A L (MyD88-adaptor like protein, also known as TIR-domain-containing adaptor domain, TIRAP), TRIF (Toll-IL-1 receptor domain-containing adaptor inducing IFN-P) and T R A M (TRIF-related adaptor molecule). Conceptually, two main pathways have been defined downstream of TLR4, one of which is MyD88-dependent pathway and the other TRIF-dependent (Fig. 5-1). It has been shown that induction of proinflammatory cytokine gene expression downstream of TLR4 requires both pathways 3 1 5 - 3 1 6 . j n contrast, activation of the transcription factor interferon 111 regulatory factor (IRF3) and the subsequent induction of IFN-P and IFN-inducible genes require only the TRIF-dependent pathway (Fig. 5-1) . M A L likely acts upstream of MyD88, since overexpression of MyD88 in embryonic fibroblasts deficient in both MyD88 and Mai was able to activate NFicB-dependent promoter activity, whereas overexpression of only Mai was not 1 4 . MyD88 has both a carboxy-terminal TIR domain and an amino-terminal death domain (DD). Through TIR-TIR and DD-DD interactions, MyD88 forms homodimers when recruited to the receptor complex 3 1 7 . The DD allows recruitment of members of the IRAK family of serine/threonine kinases (IL-1R associated kinase), which also have a DD in their amino-termini. Among the IRAK family, IRAK4 is recruited first to MyD88, followed by I R A K I . After recruitment, IRAKs undergo autophosphorylation and cross-phosphorylation and as a consequence their affinity for MyD88 decreases. This results in recruitment and activation of the adaptor TRAF6 (TNF receptor-associated factor 6). IRAKI and TRAF6 then dissociate from the receptor complex and activate TAK1 (TGF-P activated kinase 1) at the plasma membrane. ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is an adaptor protein that bridges TRAF6 to MEKK-1 (mitogen-activated protein kinase/ERK kinase kinase-1), a member of the M A P kinase kinase kinase ( M A P K K K ) (Fig. 5-1) 3 1 8 . Activation of M E K K 1 leads to subsequent phosphorylation and activation of M K K s (MAP kinase kinase), INK, and p38 3 ' 9 . The translocation of JNK and p38 to the nucleus then leads to the activation of AP-1-or ATF-2-like complexes that also regulate pro-inflammatory genes 3 1 9 . 112 TAK1 is also a member of the M A P K K K family that is essential for LPS-induced N F K B activation through the I K B kinase (IKK) complex (Fig. 5-1) . TAK1 is associated with three TAK-binding proteins (TAB): TAB1 is an activator of T A K 1 , while the other two act as adaptors for TRAF6 (TAB2 and 3) 3 2 '~ 3 2 3 . IRAKI is degraded at the membrane, and the remaining TRAF6/TAK1/TAB complex translocates to the cytosol to activate the IKK (a, p, and y) complex and M A P K pathways by phosphorylation 1 4 > 3 2 ° . The IKK complex then phosphorylates I K B leading to its ubiquitination and degradation. This allows N F K B dimers to translocate to the nucleus to drives the transcription of target genes. Activation of M A P kinase pathways as described above by TAK1 results in an early phase of M A P K phosphorylation, which is distinct from the slow phase mediated by the 315 316 TRIF-dependent pathway in MyD88 deficient mice ' . TAK1 kinase activity has been demonstrated to be required for LPS-induced p38 and JNK phosphorylation, but not for that of E R K 3 2 4 . Phosphorylation and activation of M A P K has important roles in the inflammatory response, which include histone modification that unmask binding sites for N F K B (also referred to as K B sites) in the promoters of some inflammatory cytokine genes . p38 M A P K also is involved in post-transcriptional control of cytokine mRNAs like TNF-a 3 2 6 . A member of the IFN regulatory factor (IRF) family of transcription factors, IRAF5, has been demonstrated to act downstream of TLR4-MyD88 as a master transcription factor leading to activation of genes encoding inflammatory cytokines . In macrophages and 113 dendritics cells from IRF5" mice challenged with either TLR4 or TLR9 ligands, the production of IL-12p40 was severely impaired. Levels of TNF-a and IL-6 were also significantly diminished, though not to the same extent as that of IL-12p40. IRF5 binds to MyD88 and TRAF6 directly (Fig. 5-1) and once activated translocates to the nucleus where it binds to interferon-stimulated response elements (ISRE) of IL-12p40. IRF5 is believed to cooperate with other transcription factors, because the IL-12p40 promoter has binding sites for transcription factors activated by INK and p38 M A P K , as well as K B binding sites. A similar mechanism has been proposed for IL-6 and TNF-a transcription. Although binding of IRF5 to the promoters of these cytokine genes was not examined, 197 multiple ISREs have been identified in the promoters of IL-6 and TNF-a . Recently, IRF4 has been shown to negatively regulate the production of proinflammatory cytokines (TNF-a, IL-6, IL-12p40) by macrophages in response to TLR stimulation including LPS, 198 by competing with IRF5 for MyD88 binding (Fig. 5-1) . IRF-4 is expressed in lymphocytes and macrophage and dendritic cells and negatively regulates TLR4 signaling through the MyD88 pathway, thereby attenuating the production of proinflammatory cytokines. It is important to note that the influences of IRF4 and IRF5 on cytokine production have been shown to be cell-type specific in mice. They have been observed in peritoneal and 328 splenic macrophages . Bone marrow derived macrophages and dendritic cells from either IRF4"/_ or IRF5"A mice, on the other hand, show normal secretion of inflammatory 198 cytokines compared to control cells . Therefore in different conditions or cell types, IRF5 is not an absolute requirement for LPS-induced proinflammatory cytokine 114 production, and IRF4 is not a consistent inhibitor. The roles of IRF4 and IRF5 in regulating the production of inflammatory cytokines in human monocytic cells are as yet unknown. For TLR2, TLR5, TLR7, and TLR9, activation of the MyD88 pathway alone is sufficient to result in the expression of inflammatory cytokines. In contrast, activation of the MyD88 pathway alone downstream of TLR4 results in IRAK phosphorylation and early phase N F K B activation, but these events are not sufficient to activate the expression of inflammatory cytokines 3 1 5 . In order for TLR4 to bring about the expression of 316 inflammatory cytokines, the TRIF-dependent pathway is also required . It is currently not clear why TLR4 signaling evolved to require both of these pathways 1 4 . 19Q Activation of the TRIF-dependent pathway requires the adaptor T R A M (Fig. 5-1) . 110 Following TLR4 stimulation, TRIF can recruit TRAF6 and activate N F K B and the M A P K pathways, although at a slower rate than the MyD88-dependent pathway 3 1 5> 3 1 6. Since mice deficient in both MyD88 and TRAF6 still can partially activate N F K B in response to LPS, this has led to the suggestion that TRIF must have access to at least one alternative pathway leading to N F K B activation (i.e. TRAF6-independent activation of 331 N F K B downs stream of TRIF) . The N-terminal region of TRIF can bind to both TRAF6 and TBK1 (TANK-binding kinase 1), while the C-terminus has been shown to 119 bind RIP-1 (Receptor-interacting protein-1) . Embryonic fibroblasts from RIP-1 111 deficient mice showed impaired N F K B activation in response to TLR3 ligands . Both TRAF6 and RIP-1 independently are involved in N F K B activation, although it is unclear 115 if RIP-1 mediates this in a manner similar to TRAF6. In addition, TRIF can activate TBK1, which is associated with IKKs and T A N K (TRAF-family-member-associated N F K B activator kinase) 3 3 4 . TBK1 and IKKs phosphorylate IRF3, leading to its nuclear 316 translocation and transcription of IFN-P and other IFN-inducible genes (Fig. 5-1) , . l o r T n fl Transcription of IFN-P also requires N F K B for full activation ' . Recently through the use of inhibitors of Src tyrosine kinase family, these tyrosine kinases have been shown to be required for LPS-induced IFN-P expression in the TRIF-dependent pathway 3 3 7 . How these kinases are activated by the TRIF-dependent pathway remains to be determined, however. Even in the simplified schematic diagram shown in Figure 5-1, it is evident there are at least three, parallel pathways downstream of TLR4 activation that can lead to the induction of pro-inflammatory cytokine gene expression. Furthermore, two of these lead to the transcription of IFN-related genes. The existence of these multiple pathways, the possibility of other yet to be discovered pathways combined.with post-transcriptional regulation, indicates the control of TLR4-induced cytokine production is complex and tightly regulated. PI3K and TLR Class IA PI3K has been shown to be involved in the innate immune response through 994. 99S 99R TLRs in macrophages, dendritic cells, endothelial cells, and B-cells ' ' ' . The p85 subunit has been shown to bind to phosphotyrosine residues in MyD88 in murine macrophages, and this association is significantly increased after LPS treatment. This 116 interaction with MyD88 explains how PI3K may be activated downstream of TLR4 even though the TIR domain of TLR4 does not contain a Y-x -x -M motif. This is in contrast to TLR2 or IL-1R, both of which do have Y-x-x -M motifs in their TIR domains 3 4 0 . In the context of TLR2, the PI3K p85 subunit has been shown to bind directly to TLR2 via two 987 phosphotyrosine residues . For TLR4, studies using an IRAK deficient embryonic fibroblast cell line demonstrated that IRAK is required for LPS-induced Akt phosphorylation 3 4 0 . Several mechanisms - have been proposed to explain this. These include the possibilities that IRAK may either be involved enzymatically in the activation of PI3K or its downstream effectors. Alternatively, IRAK may have a general stabilizing effect on the TLR4/MyD88/IRAK receptor complex, thus promoting downstream signaling 3 4 0 . There are two lines of evidence to suggest that PI3K is also linked to the TRIF-dependent, MyD88 independent pathway (Fig. 5-2) leading to negative regulation of the IFN-p response. In human PBMC-derived dendritic cells, wortmannin or LY294002 enhanced IFN-P expression upon TLR4 stimulation 3 4 1 . PI3K inhibitors also increased the levels of IKKcc/p phosphorylation and IKB-OC degradation with a concomitant increase in N F K B nuclear translocation. N F K B DNA-binding was also increased with PI3K inhibitors, while IRF3 DNA-binding was not affected 3 4 1 . This suggests that NFkB is required to cooperate with IRF3 to transcribe IFN-P and that PI3K inhibits the activation of N F K B but not IRF3 (Fig. 5-2). In this same study, using exogenous TRIF expression in HEK293T cells, it was demonstrated that TRIF can enhance N F K B transcriptional activity and the introduction of a catalytically active PI3K p i 10a attenuated this effect 3 4 1. 117 In this same system, p i 10a was shown to bind TRIF directly 3 4 a l t h o u g h this association has yet to be reported to occur under native conditions. Together, these results suggest, that PI3K p i 10a may be involved in attenuating TRIF-dependent activation of N F K B leading to restriction of the IFNp response. In studies concerned with TLR3 signaling in H E K 293 cells, PI3K was found to be required for full IRF3 activation 3 4 2 . In this study using H E K 293 cells expressing TLR3, PI3K was found to be necessary for a critical phosphorylation of IRF3 in order for it to be activated and bind to the ISG56 (interferon-stimulated gene 56) promoter . ISG56 is an IFN-P inducible gene involved in the inhibition of translation initiation 3 3 6 , and ISG56 promoter does not have K B sites . Furthermore, in this study it was shown that in the TLR3 pathway, TRIF acted downstream of PI3K, and that PI3K was able to bind to TLR3 via phosphotyrosine-p85 interactions. PI3K/Akt pathway was demonstrated to be essential for the serine phosphorylation of IRF3, an event necessary for IRF3 dimerization and binding to the promoter of IGF56. In this model of two-step activation of IFR3 (Fig. 5-2, right side), TRIF activated T B K l / I K K s first phosphorylates IRF3 such that it can dimerize, but not bind DNA. Subsequently, via the PI3K7Akt pathway the dimer undergoes additional serine phosphorylation, such that IRF3 can bind to D N A and interact with transcriptional coactivators such as CREB binding protein (CBP). Taken together, these results show that PI3K can participate in TRIF-dependent pathways in TLR3 signaling. It is uncertain i f this positive role for PI3K on IRF3 occurs in TLR4 signaling, or if this occurs at all in monocytic cells. 118 Taken together these two studies provide evidence that PI3K may be involved in TRIF-dependent pathways. In the context of T L R 4 signaling, PI3K serves to decrease the transcription of IFN-P by restricting N F K B activation. Figure 5-1 Adaptors Transcription factors Cytosol Nucleus Figure 5-1. Schematic diagram of T L R 4 signaling pathways. TRIF is utilized in the MyD88-independent pathway, and activates IRF3 and the subsequent induction of expression of IRF3-dependent genes such as IFN-p. IRF5 associates with MyD88 and T R A F 6 to induce inflammatory cytokine genes, while IRF4 competes with IRF5 binding 119 to MyD88 to oppose IRF5 actions. Mai and T R A M serve as bridging adaptors to recruit MyD88 and TRIF, respectively, in TLR4 signaling. TAK1 is a member of M A P K K K family, and can activate the N F K B pathway and the M A P K pathway. T A B 2/3 acts as an adaptor linking T A K to TRAF6, while TAB1 functions as an activator of T A K 1 . ECSIT interacts specifically with TRAF6 and MEKK-1 and leads to activation of M A P K pathway 3 1 8 . The two branches also modulate each other, as TAK1 can activate M K K s 319 and their downstream effectors and M E K K 1 can activate the I K K complex and N F - K B TRIF can activate the N F K B pathway through both TRAF6 and RIP-1 14>332.333>343. How RIP-1 leads to N F K B activation is not clearly understood (not shown). Not shown are dimerization of TLR4 and some adaptors, LPS complex with CD14/LBP, MD2 and other 9 1 197 198 extracellular interactions. Adapted from Ref. ' ' . 120 Figure 5-2 LPS/LBPyCD14 MAPK «t Pathways N F K B KB binding sites PRD I I S R E Figure 5-2. Model of PI3K involvement in T L R 4 - T R I F pathway and PI3K-dependent IRF3 activation in T L R 3 signaling. The left side of the figure shows the TRIF-dependent pathway downstream of T L R 4 activation. The TRIF pathway is capable 121 of mediating the late (delayed) phase of N F K B activation as well as activating IRF3. The N-terminal region of TRIF can bind to both TRAF6 and TBK1, while the C-terminus binds only RIP-1 3 3 2 . Both TRAF6 and RIP-1 are involved in N F K B activation, although it is unclear i f RIP-1 mediates this in a manner similar to TRAF6. The TBK1 pathway leads to IRF3 activation. Together, these processes lead to the transcription of IFNp. PI3K may inhibit activation of the N F K B pathway by inhibiting I K K phosphorylation, through the action of Akt, as has been previously proposed 1 9 1 . The right side of diagram shows a two step model for IRF3 activation by TLR3, proposed by Sarkar et al 3 4 2 . TRIF can recruit T B K complex and this leads to phosphorylation of IRF3 at Ser , causing IRF3 to form a dimer. This IRP3 dimer requires further phosphorylation in order to bind the promoter and to CBP more efficiently. PI3K activity provides this phosphorylation, 149 possibly via Akt . Fully activated IRF3 can then drive the transcription of ISG56. Adapted from Ref. 1 4 ' 3 3 2 ' 3 4 2 . 122 5.2 PI3K and expression of pro-inflammatory cytokines in TLR4 signaling Various studies have examined the role of PI3R in the secretion of pro-inflammatory cytokines induced by bacterial LPS through TLR4 as well as other TLR and the related IL-1R 3 4 4 , 3 4 5 . The results obtained are not entirely consistent since both positive and 00A. 00*7 negative influences have been reported " . For example, some of the evidence that focused on N F K B activation suggested a positive role for PI3K in mediating LPS signaling through TLR4. Thus, PI3K inhibitors of expression of a dominant negative of the PI3K effector Akt/PKB caused a partial defect in N F K B transcriptional activity, but not D N A binding activity 2 2 4> 3 3 9. Similarly, expression of a dominant negative mutant PI3K p85 subunit also resulted in inhibition of N F K B activity and IL-6 secretion in LPS stimulated human endothelial cells 2 2 4 . Others have reported a decrease in NO production 007 in the presence of PI3K inhibitors . Consistent with these findings, bone marrow derived macrophages (BMM) from SHIP-1"'" mice [enhance levels of PtdIns(3,4,5)P3] were reported to secrete more TNF-a, IL-6, IL-ip, and NO in response to LPS, compared to cells from SHIP-1 + / +mice 3 4 6 . The issue is somewhat muddied, however, by the fact that another report using similar cells reached opposite conclusions 3 4 7 . It has also been reported that B M M from SHIP-1 K O mice have a reduced capacity to develop tolerance to LPS, again suggesting a role for PtdIns(3,4,5)P3 in mediating inflammatory pathways 346 While the findings described above indicate that PI3K can play a positive role in regulating cytokine production, evidence to the contrary has also been reported. For 123 example, PI3K has been shown to promote IRAKI degradation in the early phase after LPS exposure 1 8 6 . PKC-^ is known to associate with I R A K I , and inhibitors of PKC prevented IRAKI degradation 3 4 8 . Since PKC-£ has been shown to be activated downstream of PI3K in LPS treated cells 1 8 8 , the IRAK/PI3K/PKC axis may provide a 186 negative feedback loop, acting to attenuate signals mediated by IRAKI . In this regard, PI3K may serve as a negative regulator of TLR4 signaling. There are other reports that suggest PI3K plays an anti-inflammatory role such as limiting the production of TNF-a and IL-12 following LPS stimulation in human monocytes and murine dendritic cells, respectively ' . An increase in NO production in the presence of PI3K inhibitors has also been reported 3 4 9 . Recently, it was shown that primary cells from SHIP"7" mice displayed an anti-inflammatory phenotype that required exposure to T G F -P and aging of the animals . In addition, it was also shown that a pathway involving PI3K mediates anti-inflammatory signaling through inhibition of glycogen synthase kinase 3p (GSK3) following LPS challenge. GSK3 is a target of Akt, and when phosphorylated it loses its ability to phosphorylate and inactivate the transcription factor cyclic A M P response element binding protein (CREB). Activated CREB then continues to sequester CREB binding protein (CBP) from N F K B , limiting activation of the latter. Consequently, secretion of IL-10 is enhanced by increased CREB activity, and proinflammatory responses including secretion of TNF and IL-6 are reduced due to suboptimal N F K B activity 124 Inconsistent data from the literature regarding whether PI3K is predominantly pro- or anti-inflammatory with respect to regulating cytokine production, may reflect species-specific or cell type-specific differences, or different in vitro conditions. Another likely possibility is that distinct PI3K isoforms regulate different pathways, some positively and others negatively. In the context of either species- or cell type-specific differences, inhibition of all PI3K isoforms using conventional inhibitors may therefore result in conflicting outcomes. A limitation common to most of the studies that investigated PI3K and TLR4 signaling was that the roles of individual PI3K isoforms were not directly assessed. An example is the use of first generation PI3K inhibitors, which also do not address individual isoform specific pathways as they inhibit all PI3K isoforms except class II PI3K C2a 4 7 ' 6 9 . These inhibitors also have been shown to be non-specific for PI3K, even at relatively low concentrations " . Another example which may have limited specificity is the SHIP knockout model, which reveals the effects of relative higher PtdIns(3,4,5)P3 levels compared to levels of PtdIns(3,4)P2. It is possible, therefore, that phenotypic changes observed in this model may reflect the effects of reduced concentrations of PtdIns(3,4)P2. ore rather than increased levels of PtdIns(3,4,5)P3 . A lack of SHIP in cells could also potentially dysregulate all of the class I PI3K pathways,, and mask contributions by individual isoforms. Given the evidence of non-redundancy in function amongst some of the pi 10 isoforms (e.g. p i 10a or p i 10P knockouts are embryonically lethal), it may not, be possible to assign a function to any isoform-specific P O K pathway through this approach alone. In the interest of overcoming some of these limitations, in this chapter, 125 we report the results of studies that investigated LPS-induced TNF-a, IL-6, IL-10, and IL-12 secretion in pi 10a deficient THP-1 cells using cytokine ELISAs, qualitative RT-PCR, and Western blot analysis. 5.3 Effects of p i 10a silencing on LPS-induced cytokine expression THP-1 cells deficient in PI3K pi 10a have an altered LPS-induced cytokine production profile. To examine the role of p i 10a on LPS-induced cytokine production, THP-1 cells deficient in p i 10a or control cells were incubated with serum opsonized LPS. Cytokine ELISAs were performed on supernatants collected at 5 h and 18 h post-exposure to LPS. THP-1 p i 10a deficient cells showed enhanced production of TNF-a (Fig. 5-3A), and IL-10 (Fig.5-3B) at both time points, compared to control cells. In contrast, LPS-induced levels of IL-12 and IL-6 were significantly diminished in p i 10a deficient cells (Fig. 5 - 3 C and Fig. 5 - 3 D , respectively). 126 Figure 5-3 A TNF-a 5h p 1 1 0 a . k n o c k d o w n shRNA control p110ctknockdown+LPS shRNA control + LPS i 1 1 1 1 • f / v / * f f pg/ml TNF-a 18h p 1 1 0 a knockdown shRNA control p 1 1 0 a knockdown+LPS shRNA control + LPS pgrml 127 Figure 5-3 B IL-10 5h pi 10a. knockdown shRNA control p110a knockdown* LPS shRNA control + LPS pgAnl IL-1018h p110ot knockdown shRNA control p110a knockdown+LPS shRNA control pgAnl 128 Figure 5-3 C IL-12p405h p110a knockodown shRNA control p110a kr»ckdown+LPS shRNA control + LPS pg/ml IL-12p4018h p110a. knockdown -shRNA control p110a knockdown+LPS shRNA control + LPS pg/ml 129 Figure 5-3 D IL-6 5h p110rx knockdown shRNA control p 1 1 0 a knockdown+LPS shRNA control + LPS IL«18tl p 1 1 0 a . knockdown shRNA control-p 1 1 0 a knockdown+LPS shRNA control + LPS # <f <f # £ Figure 5-3. Results of E L I S A for TNF-a, IL-6, IL-10, and IL-12p40 at 5h and 18h post LPS-stimulation of THP-1 cells. Cells, either transduced with control siRNA virus (shRNA control) or virus containing siRNA targeting p l lOa (pl lOa knockdown), were either incubated or not with LPS at 100 ng/ml for 5 or 18h. Error bars indicate standard deviation, n=5. A, LPS-induced TNF-a production was significantly higher in p i 10a deficient cells compared to controls at 5h (post-ANOVA Tukey test, /?<0.01) and 18h (post-ANOVA Tukey test, p<0.05). B, LPS-induced IL-10 production was significantly higher in p i 10a deficient cells compared to controls at 5h (post-ANOVA Tukey test, pO.OOl) and 18h (post-ANOVA Tukey test, /K0.001). C, LPS-induced IL-12p40 production was significantly lower in p i 10a deficient cells compared to controls at 5h (post-ANOVA Tukey test, p<0.05) and 18h (post-ANOVA Tukey test, /?<0.001), and was not significantly different from unstimulated cells (p>0.05, for both time points). D, LPS-induced IL-6 production was significantly lower in p i 10a deficient cells compared to controls at 5h (post-ANOVA Tukey test, /?<0.05) and 18h (post-ANOVA Tukey test, /K0.001), and was not significantly different from unstimulated cells (p>0.05, for both time points). 131 LPS-induced cytokine production in THP-1 cells is mediated through TLR-4. To verify that in this model LPS was signaling through TLR-4, THP-1 cells were preincubated for 1 h at 37°C with neutralizing antibodies to either TLR-4 or TLR-2, prior to LPS stimulation. At 18h post-LPS exposure, IL-12p40 or IL-6 ELISAs were performed on the supernatants (Fig. 5-4). Pre-incubation with anti-TLR-4 antibody reduced IL-12p40 and IL-6 production by 54% f><0.001) and 76% (p<0.0\), respectively, relative to preincubation with anti-TLR-2 antibody (Fig. 5-4A and 5-4B). The quantities of LPS-induced IL-12p40 and IL-6 production in the presence of anti-TLR2 antibody were not significantly different from LPS-stimulated control cells incubated in the absence of antibody (+LPS, j?>0.05). In contrast, preincubation with anti-TLR-2 antibody only diminished zymosan-induced TL-12p40 (44%, p<0.01), but not IL-6 (p>0.05) production relative to preincubation with anti-TLR4 antibody, suggesting zymosan might trigger IL-6 production through other receptors as well. The amounts of IL-12p40 and IL-6 produced in response to zymosan in the presence of anti-TLR4 antibody were not significantly different from those produced by zymosan-treated control cells without neutralizing antibody preincubation (+Zy, p>0.05). 132 Figure 5 - 4 A IL-12p40 aTLR4 + Zy aTLR2 + Zy + Zy aTLR4 + LPS aTLR2 + LPS + LPS - LPS -I B aTLR4 + Zy aTLR2 + Zy + Zy aTLR4 + LPS aTLR2 + LPS + LPS -LPS pg/ml 133 Figure 5-4. Effect of neutralizing antibodies to TLR-4 and TLR-2 on LPS and zymosan induced IL-12p40 and IL-6 production in control shRNA transduced THP-1 cells. A, IL-12p40 and B, IL-6 ELISA were performed on supernatants derived from THP-1 cells that had been transduced with control siRNA virus, preincubated for 5h with either TLR-4 (aTLR4) or TLR-2 (aTLR2) neutralizing antibody, and then incubated with LPS (100 ng/ml) or serum opsonized zymosan for 18h. Pre-incubation with artti-TLR-4 antibody reduced IL-12p40 and IL-6 production by 54% (post-ANOVA Tukey test /?<0.001) and 76% (post-ANOVA Tukey test /?<0.01), respectively, relative to preincubation with anti-TLR-2 antibody. In contrast, preincubation with anti-TLR-2 antibody only diminished zymosan-induced IL-12p40 (44%, post-ANOVA Tukey test /?<0.01), but not IL-6 (post-ANOVA Tukey test />>0.05) production. There was no significant difference between LPS stimulation alone vs anti-TLR2 with LPS, or between zymosan alone and anti-TLR4 plus zymosan (/?>0.05). Error bars indicate standard deviation. The data are representative of two independent experiments. 134 The presence of neutralizing antibodies to TNF-a or IL-10 did not normalize the cytokine response of pi 10a deficient THP-1 cells. Exogenous TNF-a has been reported to augment IL-10 production in monocytes . We considered the possibility that enhanced TNF-a production by p i 10a deficient cells might bring about augmented IL-10 production via a paracrine effect. To examine this possibility, p i 10a deficient THP-1 cells were preincubated or not with anti-TNF-a or isotype-matched control antibodies. Figure 5-5 shows that anti-TNF-a neutralizing antibodies restored neither LPS-induced IL-10 (Fig. 5 - 5 B ) , IL-12p40 (Fig. 5 - 5 C ) , nor IL-6 (Fig. 5 - 5 D ) production by p i 10a deficient cells to control levels. However, anti-TNF-a antibodies did moderately suppress IL-6 production in p i 10a deficient cells compared to isotype matched controls (Fig. 5 - 5 D and 5 - 6 B ) . OCT IL-10 has also been reported to suppress LPS-induced IL-6 and ILT12 production . To investigate whether diminished LPS-induced IL-6 and IL-12 secretion by p i 10a deficient THP-1 cells was due to enhanced IL-10 production, neutralizing antibody to IL-10 was added or not prior to stimulation of cell with LPS (Fig. 5 - 5 C and 5 - 5 D ) . Preincubation with neutralizing anti-IL-10 antibody did not restore IL-6 or IL-12 production to levels seen in control cells. Finally, coincubation of cells with both anti-TNF-a and anti-IL-10 neutralizing antibodies together also did not restore IL-6 or IL-12 to control levels (Fig. 5 - 6 A and 5 - 6 B , respectively), ruling out a possible synergism between enhanced TNF-a and IL-10 levels in the suppression of IL-12 and IL-6 production by p i 10a deficient cells. 135 Figure 5-5 A TNF-a. 9 l shRNA cmftnl +LPS No Tx shRNA cortroi 40ugftnl al-10 + LPS 20ugftnl al_-10 + LPS lOugfrnl al_-10 + LPS 20ugftnl riGg + LPS 40ug/ml aTNFa + LPS 20ugAid aTNFa t LPS 10ug/ml aTNF<z+ LPS 20ugfml mlgG + LPS LPS NoTx ^ ^ ^ ^o ^ ^ ^ j ^ j j ^ / * ^ B 1.-1019h s h R N A con t ro l + L P S N o T x s h R N A G U M iOttgim a l , 1 0 * L P S 2 0 u g A r f a H O * L P S lOugf t r t a t 10 • L P S 20u t jAn i ricjG * L P S 4 0 u g f m l a T N F a * L P S 2 0 u g A n I a T W a + L P S l O u p l h i l a T N F a + L P S 20ugf tn l m l g G • L P S L P S N D T X i 1 1 1 1 r -pgTiii 136 Figure 5-5 C L - 1 2 p 4 0 1 9 h shRNA control • LPS No Tx shRNA control 40uglhilaL-10 • LPS 20ugftnial_-10 • LPS 10ug*nlal_-10 •LPS 2DuglhililGg • LPS 40ugAnl aTNFtx • 1 PS 20ugAnl aTNFa • LPS lOugftnl aTNFa • LPS 20ugftnlmlgG • LPS LPS NoTx D shRNA conbol +LPS No Tx shRNA control 40ugftnl ai_-10 • LPS 20ugftni a t 10 • LPS lOutjAni a t 10 •LPS 20ugAnlitGg + LPS 4flugAiri aTNFa* LPS 20ugAnl aTNFa * LPS lOugAnl aTNFa + LPS 20ugAi«l tnlcjG • LPS LPS No Ix ^ 4? pgftnl L-G19h pa/ml 137 Figure 5-5. Effect of neutralizing anti-TNF-a or anti-IL-10 antibodies on LPS-induced IL-12p40 and IL-6 production by THP-1 cells. THP-1 cells transduced with control siRNA virus (shRNA control, clear bar graphs) or virus containing siRNA targeting p i 10a (pi 10a knockdown, filled bar graphs) were preincubated or not with anti-TNF-a antibody (aTNFa), anti-IL-10 (aIL-10), or isotype matched control antibody (mlgG or rlgG) at the indicated concentration for 5h. Cells were then either stimulated or not (No Tx) with LPS at 100 ng/ml for 5 or 18h. ELISAs for TNF-a (A), IL-10 (B), IL-12p40 (Q, or IL-6 (D) were then performed on supernatants. For A and B, samples treated with neutralizing antibodies TNF-a (A) or IL-10 (B) were all significantly reduced compared to control antibody plus LPS (post-ANOVA Tukey test p<0.00l). For C and D, shRNA control cells stimulated with LPS was significantly higher than all other treatment groups (post-ANOVA Tukey test p<0.00l), while the remaining groups were not significantly different from each other (post-ANOVA Tukey test p>0.05). Error bars indicate standard, deviation. The data are representative of two independent experiments. 138 Figure 5-6 A shRNA control + LPS (-) shRNA control -| rlgG-HrigG+LPS IgG ctl + LPS aTNFa-HUL-10+LPS alL-10 + LPS aTNFa + LPS + LPS IL-12p4019hr H i 1 r i 1 1 1 1 • C N." \T V NT ^ NT \ . shRNA control +LPS (-) shRNA control-| rigG + mkjG +LPS IgG cU + LPS aTNFa + aIL-10 +LPS alL-10 + LPS aTNFa + LPS + LPS H IL-12p40 48hr * # # & £ 4? pg/ml Ay 139 Figure 5-6 B shRNA control + LPS {-) shRNA control -\\ rigG + mJgG +LPS IgG + LPS aTNFa -HUL-10+LPS alL-10 + LPS aTNFa + LPS + LPS ft IL-6 19hr 4^ ft pgrm! IL-648hr shRNA corrtrol + LPS (-) shRNA control rigG + mlgG +LPS IgG ctJ + LPS aTNFa +3IL-10+LPS alL-10 + LPS aTNFa + LPS + LPS {-) pg/mi 140 Figure 5-6. Coincubation of neutralizing anti-TNF-a and anti-IL-10 antibodies did not restore LPS-induced IL-12p40 and IL-6 production. THP-1 cells transduced with control siRNA virus (shRNA control, clear bar graphs) or virus containing siRNA targeting p i 10a (pi 10a knockdown, fdled bar graphs) were preincubated or not with anti-TNF-a antibody (aTNFa), anti-IL-10 (aIL-10) antibody, or isotype matched control antibodies (mlgG, IgG) at the indicated doses for 5h. The cells were then either stimulated or not (-) with LPS at 100 ng/ml for 19 or 48h. ELISAs for IL-12p40 (A) or IL-6 (B) were then performed on supernatants. Error bars indicate standard deviation. The data are representative of two independent experiments. 141 5.4 Activation of signaling pathways by p i 10a in response to LPS Activation of N F K B and p38 M A P kinase have been shown to be important in the regulation of LPS-induced cytokines such as TNF-a, IL-6 and IL-12 1 9 I> 2 2 8, LPS is known to activate all three M A P kinases p38, INK, and E R K 3 5 8 . Since LPS stimulated p i 10a deficient,cells had an enhanced ability to secrete TNF-a but diminished capacity to secrete IL-6 and IL-12, we examined the phosphorylation status of M A P kinases, as well as that of N F K B p65. In addition, to determine if the changes in cytokine profde observed were due to transcriptional or post-transcriptional regulation, semi-quantitative RT-PCR of cytokine mRNA was examined. 5.4.1 Western blot analysis LPS-induced phosphorylation of p38, but not JNK or ERK, is enhanced in pi 10a deficient THP-1 cells. It has been reported that LY294002 pretreated monocytic cells have enhanced LPS-induced TNF-a production and this correlates with increased phosphorylation of p38, ERK, and JNK kinases 1 9 1 . We carried out Western blot analyses to determine if these changes would be observed in p i 10a deficient THP-1 cells. Cells transduced with shRNA against p i 10a were markedly deficient in pi 10a as compared to control shRNA transduced cells (Fig. 5 - 7 A ) . Of interest, anti-phospho-Akt antibodies revealed that whereas LPS treatment resulted in increased phosphorylation of Akt in control cells, no significant increase in phospho-Akt was observed in pi 10a deficient cells (Fig, 5 - 7 B ) . Since Akt is activated as a downstream target of PI3K, these findings, suggest that the 142 only PI3K responsible ,for bringing about Akt phosphorylation—indirectly through PDK1—in response to LPS cell treatment is p85/pl 10a. We also observed that phosphorylation of p38 was enhanced in p i 10a deficient cells after 20 and 30 min post-LPS stimulation compared to control cells. In contrast, the phosphorylation of neither JNK (Fig. 5-7B) nor E R K was significantly altered in p i 10a deficient cells when compared to controls and after normalization for protein loading (Fig. 5-7C). On the other hand, the results shown in Figure 5-7D indicate that like p38, phosphorylation of N F K B p65 was also clearly enhanced in p i 10a deficient THP-1 cells relative to controls. 143 Figure 5 -7 P110a « • *# Actin — —• B fi fi Phospho-Akt Phospho-JNK Phospho-p38 GAPDH LPS • **m mm. "''SHU ^ "*^ ! S J ' ^ • W •mm- • mm - 5 10 20 30 60 - 5 10 20 30 60 Phospho-ERK GAPDH PI3K p110a1 PI3K p110a3 LPS 20 30 PI3K p110a1 5 20 30 PI3K p110a3 Phospho-p65/NFKB GAPDH LPS mm ~*# ««. *•* #** ««•» ... — - 5 20 30 - 5 20 30 PI3K p110a1 PI3K p110a3 Figure 5-7. Western blot analysis. Antibodies to: A, PI3K p i 10a, B, phospho-Akt, phospho-JNK, phospho-p38, C, phospho-ERK, and D, phospho-p65 N F K B , in THP-1 144 cells that were either transduced with control siRNA virus (PDKpl lOa l ) or virus containing siRNA targeting p i 10a (POKpl 10a3). Cells were either incubated or not (-) with LPS at 100 ng/ml at various times (minutes). G A P D H was used as protein loading control. Anti-phospho-p65 N F K B antibody was assessed on total cell lysates. ,Results are representative of three independent experiments. 145 5.4.2 mRNA levels for TNF-a, IL-6, IL-10, and IL-12 Using RT-PCR, we examined whether the changes observed in LPS-induced cytokine expression in p i 10a deficient cells could be explained by corresponding alterations in mRNA levels (Fig.5-8). Consistent with reports that TNF-a is regulated both transcriptionally and post-transcriptionally ' , TNF-a mRNA levels from cells deficient in p i 10a were not significantly enhanced compared to controls, despite the fact that these cells produced more TNF-a (Fig. 5 -3A) . Likewise increased secretion of IL-10 by pi 10a deficient cells was not reflected by increased IL-10 mRNA levels after LPS-stimulation. In contrast, IL-12p40 mRNA levels in p i 10a deficient cells were significantly less than those of control cells, particularly at 4h post-LPS stimulation. This was also found to be the case for IL-6 where mRNA levels were lower in p i 10a deficient cells when compared to control cells at 4 and 16h. These results suggest that diminished IL-12 and IL-6 production by p i 10a deficient cells may in part be explained by events at the level of mRNA accumulation, but this does not appear to be the case for enhanced TNF-a and IL-10 production. 146 Figure 5-8 Figure 5-8. RT-PCR for TNF-a, IL-12p40, IL-6, and IL-10, mRNA in LPS-stimulated THP-1 cells deficient in POK pi 10a. THP-1 cells either transduced with control s i R N A virus ( a l ) or virus containing s i R N A targeting p i 10a (a3) were stimulated with 100 ng/ml L P S (+) or not (-) for the times indicated. Cells were then collected and c D N A were generated from the R N A preparations. Act in was used as loading control. Results are representative of three independent experiments. 147 5.5 NADPH-dependent oxidase and PI3K Professional phagocytes have critical roles in innate immunity and the production of microbicidal oxidants is a key component of host defense. This is exemplified by a genetic disease in humans called chronic granulomatous disease (CGD), characterized by severe recurrent bacterial and fungal infections 2 3 0 . The generation of microbicidal oxidants by monocytes and neutrophils requires the activation of a multi-protein complex called the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Genetic defects in CGD patients result in either absent or defective components of the N A D P H oxidase 2 3 0 . Cells from these patients are normal in terms of phagocytosis, chemotaxis, and degranulation, but are unable to generate an oxidative burst 3 6 ° . 5.5.1 Assembly of the NADPH-dependent oxidase complex is highly regulated and requires P O K activity The regulation of N A D P H oxidase function is tightly regulated, as unregulated production of reactive oxygen species (ROS) can result in deleterious effects on the host . Control is achieved through both spatial and temporal regulation of the N A D P H oxidase complex assembly and activation . The multi-component N A D P H oxidase is unassembled and inactive in resting cells^ but assembles at the plasma or phagosomal membrane upon phagocyte activation (Fig. 5-9A) . The N A D P H oxidase complex consists of two membrane-bound components (gp91 p h o x and p22 p h o x), and at least four cytosolic components (p40 p h o x, p47 p h o x , p67 p h o x , and Rac GTPase) 3 6 1 . The phox family of proteins is named for phagocyte oxidase . The N A D P H oxidase in human 148 1 £Ti monocytes uses Racl , while neutrophils use Rac2 . The membrane bound components form a heterodimer called flavocytochrome bsss, which contains all the redox components 361 used by the N A D P H oxidase for transmembrane electron transport Assembly of the NADPH-dependent oxidase requires PI3K activity and 3'-phosphoinositides 3 6 0> 3 6 4. PI3K serves at least two important roles in N A D P H oxidase activation including recruitment and localization of p47 p h o x and p40 p h o x to the membrane, and activation of p47 p h o x . In this regard, phagosomal lipid rafts have been proposed to be platforms for the recruitment of cytosolic N A D P H oxidase components and for their assembly into an active N A D P H oxidase complex ' . Binding or phagocytosis of particulate agonists such as opsonized zymosan (OPZ) to CR3 leads to activation of class IA PI3K via Src family phosphotyrosine kinases such as Hck ' \ . The PI3K product PtdIns(3,4,5)P3 generated on the plasma membrane is then able to recruit PH domain containing effectors such as PDK1 and P L C v 2 ' 1 9 0 , leading to activation of Akt and PKC, respectively (Fig. 5 - 9 A ) . Depending on the agonist, a number of kinases have been proposed to phosphorylate p47 p h o x , an event that is critical in the activation of N A D P H oxidase assembly. These kinases include PKC 1 9 0 , M A P K p3 8 2 3 1 , Erkl/2 3 6 9 , 1 7 0 171 1 7 7 ™» p21 activated kinase (PAK) , Akt , and phosphatide acid-activated kinase . The most convincing physiological evidence has been shown for P K C , while the physiological roles of the other kinases are less well understood 3 6 1 . Both classical PKC (a, p i , pil) and novel P K C isoform (5) have been reported to mediate p47 p h o x phosphorylation 1 9 ° . Soluble agonists like phorbol esters (PMA) can activate cPKC and nPKC. Activated P K C is then able to phosphorylate critical serine residues on p47 p h o x , 149 which in the unstimulated state assumes an inactive intramolecular conformation (Fig. 5-9A) 3 6 0 . Akt has also been reported to phosphorylate p47 p h o x in a cell-free system derived from neutrophils 3 7 3 , but experiments in monocytic cells using Akt inhibitors show that fMLP-induced N A D P H activation is independent of Akt 1 9 0 . It is not clear whether this also applies to the OPZ-induced oxidative burst. Once phosphorylated by PKC, a conformational change results which exposes the various domains of p47 p h o x such as the PX and SH3 domains (Fig. 5-9A) 3 6 0 . Phosphorylated p 4 7 P h o x i g m e n a b k t Q i n t e r a c t w i t h p 6 7 P h 0 X ; p 4 0 p h o x , and p22 p h o x through interactions with their SH3 and PxxP (proline-rich motif) domains (Fig. 5-9A) . The two cytosolic components, p40 p h o x and p47 p h o x , contain a Phox homology (PX) domain 3 7 4 . P X domains are known to bind various phosphoinositides 160>162>364, and the P X domain of p40 p h o x specifically binds PtdIns(3)P 3 7 4 , 3 7 5 , while the P X domain of p47 p h o x specifically binds PtdIns(3,4)P2 3 7 4 . Studies have identified two lipid phosphatases, SHIP-1 and PtdIns(3,4)P2 4-phosphatase, that are required to metabolize class I PI3K product PtdIns(3,4,5)P3 in order to recruit cytosolic oxidase components to the membrane [reviewed in Ref. 3 6 4>3 7 6]. SHIP-1 removes the 5' phosphate from PtdIns(3,4,5)P3 to generate Ptdlns(3,4)2, and the.latter recruits p 4 7 P h o x 3 7 5 . Subsequently, the PtdIns(3,4)P2 4- phosphatase can generate PtdIns(3)P from Ptdlns(3,4)2 . This results in the recruitment of p40 p h o x to the membrane. Other interactions between the membrane components (gp91 p h o x, p22 p h o x , Racl) and the cytosolic components are shown in Figure 5- 8A. Oxidase activity requires p67 p h o x to associate directly with flavocytochrome 055$ 150 (gp91 p h o7p22 p h o x) and this has been proposed to mediate allosteric effects on the catalytic activity of flavocytochrome bssz Rac translocation to the membrane is also an essential element of N A D P H oxidase activation 3 6 1 . In the basal state, Rac is associated with RhoGDI (Rho GDP dissociation inhibitor) in an inactive GDP-bound form. Upon phagocyte activation, Rac dissociates from RhoGDI and exchanges GDP for GTP and translocates to the N A D P H oxidase complex at the membrane. Active Rac can then bind the TPR (tetratricopeptide repeat) domain of p67 p l l 0 X and interact directly with gp91 p h o x . PI3K may activate Rac through activation of Vav, a PH-domain containing Rac-GEF 2 3 6> 3 7 7. However, in OPZ-stimulated bovine neutrophils, M A P K p38 has also been shown to activate Rac and promote its n no translocation to the membrane, and p38 activation was PI3K dependent . Yamamori et al proposed that p38 can activate cytosolic phospholipase A 2 (cPLA 2 ) , leading to the release of arachidonic acid . Arachidonic acid is then able to induce the,dissociation of Rac and RhoGDI, resulting in Rac activation . Further studies will be required to clarify the exact mechanism of Rac activation. Overall, Rac, p47phox and p40 p h o x seem to function as adaptors to provide a stable platform for the interaction between the catalytic component (gp91 p h o x and p22 p h o x) and the regulatory component (p67 p h o x) of the N A D P H oxidase 3 6 0 . Once the oxidase is assembled at the membrane, electrons are transferred from N A D P H in the cytoplasm through the gp91 p h o x component across the membrane leading to the reduction of intra-phagosomal or extracellular 0 2 to superoxide anion 0 2" (Fig. 5-9B). Protons that exit the 151 cytosol via voltage-gated proton channels are then able to react with O2" to generate hydrogen peroxide (H2O2). This can occur either spontaneously or from the action of superoxide dismutase (SOD). H2O2 may be converted to hypochlorous acid (HOC1) by myeloperoxidase (MPO). MPO is expressed in monocytes and neutrophils , but not normally expressed in murine macrophages and cultured human macrophages Termination of N A D P H oxidase activity is less well understood, but it correlates with a loss of p47/67 p h o x from the membrane 2 4 3 , and phosphorylation of p47 p h o x by casein 381 kinase 2 has been shown to deactivate the oxidase 152 NADP + + 2H + Figure 5-9. Model of N A D P H oxidase assembly and activation. A, Assembly of the NADPH-dependent oxidase requires PI3K activity and 3'-phosphoinositides. Binding of 153 particulate agonists such as opsonized zymosan (OPZ) to CR3 leads to activation of class . IA PI3K via Src family phosphotyrosine kinases (PTK), possibly Hck ' ' . PtdIns(3,4,5)P3 generated on the plasma membrane is able to recruit PH domain containing effectors such as PDK1 and PLCy2, leading to activation of Akt and PKC, respectively. Both classical P K C (cPKC) and novel P K C isoform (nPKC) have been reported to mediate p47 p h o x phosphorylation 1 9 0 . Soluble agonists like P M A can activate both cPKC and nPKC. In the unstimulated state, p47 p h o x forms an intramolecular complex. Activated P K C is able to phosphorylate serine residues on p47 p h o x and induce a conformational change which exposes the various domains of p47 p h o x thereby facilitating interactions with other proteins. The role of Akt in p47 p h o x phosphorylation in vivo is uncertain. Rho GTPase has also been reported to be essential for p47 p h o x phosphorylation downstream of OPZ stimulation 3 8 2 , although the mechanism of this is not clear. Phosphorylated p47 p h o x is then able to interact with p67 p h o x , p40 p h o x , and p22 p h o x through various SH3-PxxP (proline-rich motif) interactions (shown by red bidirectional arrows). SHIP-1 removes the 5' phosphate from PtdIns(3,4,5)P3 to generate Ptdlns(3,4)2, which then recruits p47 p h o x . The PtdIns(3,4)P2 4-phosphatase can generate PtdIns(3)P from Ptdlns(3,4)2. This results in the recruitment of p40 p h o x to the membrane as well. Other interactions between the membrane components (gp91 p h o x, p22 p h o x , Racl) and the cytosolic components are shown in biack bidirectional arrows. Rac is associated with RhoGDI (Rho GDP dissociation inhibitor) in the inactive GDP-bound form. Upon phagocyte activation, Rac dissociates from RhoGDI and exchanges GDP for GTP and translocates to the N A D P H oxidase complex at the membrane. Active Rac can bind TPR (tetratricopeptide repeat) domain of p67 p h o x and interact directly with gp91 p h o x . PI3K 154 'J'IC 177 may active Rac through activation of Vav, a PH-domain containing Rac-GEF ' . p38 M A P K may also regulate the dissociation of Rac/RhoGDI complex via arachidonic acid (AA) by activation of c P L A 2 3 7 8 . Not shown are the interactions between the three cytosolic components (p40 p h o x, p47 p h o x , and p67 p h o x) in the resting state, and the contribution of class IB PI3K downstream of GPCR (e.g. fMLP receptor). Adapted from ref. 1 9 0 > 2 3 6 . 3 6 °. 3 6 4 > 3 7 6 g, Generation of ROS in phagosomes contributes to microbicidal activity. Electrons are removed from N A D P H in the cytosol and transferred through the gp91 p h o x component across the membrane to reduce intraphagosomal/extracellular molecular 0 2 to 02". Protons exit the cytosol via voltage-gated proton channels. Hydrogen peroxide (H 2 0 2 ) is generated from 0 2" and H + either spontaneously or through the action of superoxide dismutase (SOD). H 2 0 2 may be converted to hypochlorous acid (HOC1) by myeloperoxidase (MPO). Adapted from Ref. 3 9 . 155 5.5.2 Effect of p i 10a silencing on activation of the phagocyte oxidase by P M A and opsonized zymosan To examine the role of p i 10a in the oxidative burst, both soluble and particulate agonists were used. THP-1 cells deficient in p i 10a or transduced with control shRNA were differentiated with low dose P M A (1.6 nM) for 20 hr, and rested in fresh media for 4 hr before stimulation by either high dose P M A (1 pM), serum opsonized zymosan (OPZ), or unopsonized zymosan (Z) (Fig. 5-10). P M A induced robust superoxide production in control cells, while cells deficient in pi 10a were significantly impaired (Fig. 5-10A) with a mean reduction of 76% (/?<0.001). For OPZ, control cells similarly produced significant superoxide compared to unstimulated cells (p<0.00\). In contrast, OPZ- • stimulated THP-1 cells deficient in p i 10a had a significant reduction in superoxide production (Fig. 5-10B) with a mean decrease of 54% (p<0.0\). In contrast to either P M A or OPZ, no significant production of superoxide was detected in control cells when stimulated by unopsonized zymosan (Fig. 5-10C). These results indicate that pi 10a is required for superoxide production in response to both the soluble agonist P M A and the particulate agonist OPZ. In this system, non-opsonized zymosan did not appear to bring about oxidase activation. 156 Figure 5-10 A B 7.5 7.5-i No agonist Zym No agonist Zym shRNA control p110a knockdown Figure 5-10. p85/pll0a PI3K is required for P M A - and opsonized zymosan-induced oxidase activation. THP-1 cells deficient in p i 10a or transduced with control shRNA were differentiated overnight with 10 ng/ml (1.6 nM) of P M A , and rested in R P M I for 4h prior to stimulation. A, PMA- induced superoxide production. THP-1 cells were stimulated or not with 1 p M P M A for 30 min. In response to P M A stimulation, cells 157 deficient in pi 10a were significantly reduced in superoxide production compared to control cells (post-ANOVA Tukey test, pO.OOl). Unstimulated control cells were not significantly different from stimulated pi 10a deficient THP-1 cells (post-ANOVA Tukey test, p>0.05), «=11. B, Opsonized zymosan (OPZ) induced superoxide production. Zymosan particles were opsonized with human serum for 30 min at 37°C prior to use. The ratio of particles to cells was 20:1. In response to OPZ, cells deficient in pi 10a were significantly reduced in superoxide production compared to control cells (post-ANOVA' Tukey test, pO.Ol). Unstimulated cells were not significantly different from OPZ-stimulated pi 10a deficient THP-1 cells (post-ANOVA Tukey test, p>0.05), n=l. \ C, Unopsonized zymosan (Z) did not induce superoxide production significantly in THP-1 cells (one-way ANOVA, p=0.3437). Cells were stimulated or not by zymosan particles at a ratio of 20:1, n=3. Data are expressed in nanomoles of O2" produced by 0.5 x 106 cells in 30 min. A p-value <0.05 was considered significant. Error bars indicate SEM. 158 5.6 Discussion The data presented here suggest that the p85/110a isoform of PI3K positively regulates the production of the proinflammatory cytokines IL-12 and IL-6, and concurrently negatively regulates the production of TNF-a and a major anti-inflammatory cytokine IL-10 in response to LPS. In control THP-1 cells, LPS-induced TNF-a rose rapidly and reached a maximum by five hours (Fig. 5-3A), while IL-6, IL-12p40, and IL-10 rose more slowly reaching plateaus between 19h to 48h post LPS stimulation (Fig. 5-6A to C). These dynamics of cytokine production in response to LPS were consistent with what has ' 356 357 been reported previously for human monocytic cells ' . It has been shown previously that the use of PI3K inhibitors resulted in augmented production of TNF-a by LPS-treated human monocytic cells 1 9 1 . However, because LY294002 and wortmannin are neither P O K class- nor isoform-specific, it has not been possible to assign this function to an individual POK. The results of the present study, show for the first time in human monocytic cells that an individual isoform of POK—in this case p85/pll0a—is able to regulate the production of TNF-a and other cytokines. This suggests that the other class IA isoforms have non-redundant functions at least in terms of this cellular response to LPS. In the report by Guha and Mackman referred to above where biochemical inhibitors of P O K kinase were used, enhanced production of TNF-a correlated with enhanced p38 phosphorylation and concurrently increased in N F K B activation and binding. These investigators proposed that P O K serves to limit the production of inflammatory cytokines 159 such as TNF-a, so that its expression is only transient in nature 1 9 1 . We now present data to show that this pathway appears to be regulated by the p i 10a isoform of PI3K since phosphorylation of N F K B p65 was clearly enhanced in p l lOa deficient THP-1 cells relative to controls ( F i g . 5-7C). In addition, we also found that activation of p38 M A P K in response to LPS was clearly under the control of p85/pll0a. Unlike the results obtained using PI3K inhibitors 1 9 1 , however, we did not find that the phosphorylation of JNK or E R K were significantly enhanced relative to controls ( F i g . 5-7A and B ) . Consistent with our findings, however, DN-p85 or DN-Akt had no effect on LPS-induced JNK activation in human endothelial cells 2 2 4 . Differences between p85/pl lOa-deficient THP-1 cells and those obtained using non-selective inhibitors 1 9 1 may reflect other p i 10 isoform-specific effects, differences in cell-type, or alternatively non-specific effects of inhibitors. Although we did not assess mRNA stability, results of semi-quantitative RT-PCR ( F i g . 5-8) suggested that the early augmentation of LPS-induced TNF-a secretion in pi 10a deficient cells ( F i g . 5-3) was likely due to a mechanism unrelated to mRNA accumulation and was likely post-transcriptional. In fact, post-transcriptional regulation of TNF-a production in murine splenic cells has been reported to be positively regulated by p38 M A P K . This is consistent with our finding of enhanced phosphorylation of p38 M A P K in LPS-treated p i 10a deficient cells ( F i g . 5-7B). LPS-induced TNF-a production by murine splenic cells has also been shown to be regulated post-326 '3 83 transcriptionally ' . Kotlyarov et al demonstrated that the mechanism of translational control of LPS-induced TNF-a production involved the p38 substrate MAP-kinase-160 activated protein kinase-2 ( M A P K A P - K 2 , or M K 2 ) . The mechanism involved activation of the p38 pathway which contributed to upregulation of cytokine genes at multiple levels ' . p38 and other signaling pathways can regulate cytokine genes at the transcriptional level through activation of transcription factors . In addition, regulation of cytokine translation by the p38 pathway is mediated by >a downstream kinase, M K 2 . M K 2 can phosphorylate proteins that bind to A U - r i c h elements (ARE) located in the 3'-untranslated region of cytokine m R N A s to regulate cytokine translation 3 8 3 . Phosphorylation of these ARE-binding proteins results in the release of translational repression of T N F - a m R N A . Consistent with this model, IL-10 has been shown to negatively regulate T N F - a by inhibiting T N F - a translation, and this action involves inhibition of the p38 /MK2 pathway 3 8 4 . Therefore, the increased p38 phosphorylation we observed may contribute to enhanced T N F - a and IL-10 production in pllObc deficient cells, despite the findings that m R N A levels for these cytokines were not significantly elevated compared to control cells (Fig. 5-8). Studies in knockout mice had previously suggested that PI3K can regulate cytokine production, but the isoforms involved were not clearly identified. Thus, Fukao et al demonstrated that LPS-induced IL-12 production was enhanced in dendritic cells isolated 998 form p85a K O mice . In addition, when enteric bacteria from the ceca of control mice were inoculated into the peritoneal cavities of p85a K O mice, the peritoneal washings i oi showed diminished T N F - a production compared to cells from wild-type mice Notably, these findings are the converse of those we described above, where we found that p 8 5 / p l l 0 a negatively regulated T N F - a and positively regulated IL-12. The 161 observation that bacteria-induced TNF-a production was diminished in the p85a K O mice is also the converse of what Ghua and Mackman's found when they examined TNF-a production in response to LPS in human monocytic cells pretreated with LY294002 to inhibit PI3K 1 9 1 . Several possibilities may account for these differences. First, the wild-228 type DC used in the murine model did not express p i 10a or p i 105, but only p i 10(3 . I t is conceivable that the absence of both p i 105 and p i 10a from these cells might have contributed to different outcomes. Second, cells from the p85a K O mice were not uniform in their expression of either the regulatory subunits or the catalytic subunits of PI3K, and this varied with cell type. For example, while the catalytic subunits p i 10a and pi 105 were absent from bone marrow derived-dendritic cells (BMDC), bone marrow derived mast cells (BMMC) were only deficient in pi 10a and they were normal for other 183 228 isoforms ' . Moreover, p55a and p50a regulatory subunits were found to be increased in T-cells and adipocytes, but normal in B M M C and B M D C 1 8 3 - 2 2 8 . An additional major concern about these cells from knockout mice is that they were derived from animals that had been reported by numerous groups to exhibit increased PI3K activity despite being null for p85a. For example, PI3K signaling downstream of insulin receptor is known to be dependent on class IA PI3K 1 8 4 . Based upon this, it would be predicted that a true P O K K O would exhibit a diabetic phenotype. Surprisingly, P O K signaling was enhanced and sustained for a longer time in p85a K O mice than in wild-type mice, and as a result these p85a K O mice were hypoglycemic 1 1 0 ' 1 1 3 . Moreover, heterozygous disruptions of either p85a or p85p knockout mice were observed to have improved insulin signaling m ' 1 1 2 . Taken together these results indicate that p85a knockouts cannot be considered as null for class IA POK. 162 Okkenhaug and Vanhaesebroeck suggested that defective regulation of catalytic subunits due to the absence of regulatory subunits in p85 knockout cells likely contributes to. enhanced signaling in some contexts and diminished PI3K signaling in others ! 7 4 . One example of this is the finding in p85a K O cells of increased PI3K activity associated with the p85(3 isoform, and this may account for the paradoxical observation of increased PI3K signaling in p85a K O cells 1 1 S i n c e p85 normally inhibits the kinase activity of its associated p i 10 subunit in the basal state, disruption of p85 could result in hyperactive p i 10 subunits U 2 ' 1 7 4 . To return to the issue of addressing how our results diverge from those of Fukao et al, it is important to note that in their murine, dendritic cell p85a K O model Akt phosphorylation was only mildly diminished following LPS stimulation. This indicates that significant class IA PI3K activity was still present in these p85oc K O dendritic cells 6 0 . 998 Thus, the simplest way to reconcile the reports of enhanced IL-12 and reduced TNF-a 1 81 production by cells from p85a knockout mice with our converse findings is to conclude that these mice were not true class IA PI3K knockouts. On the other hand, the superiority of using siRNA to selectively silence the class IA isoform p i 10a was amply shown based upon two important criteria: (1) deficiency of p i 10a expression while preserving normal expression of residual catalytic and regulatory isoforms (Fig. 3-3), and (2) demonstration of a bona fide and unambiguous defect in PI3K signaling (Fig. 5-7) 2 4 4 . Whether or not other variables such as species-specificity or differences in experimental 163 models also may have contributed to these contrasting phenotypes remains to be determined. Exogenous TNF-a has been reported to augment IL-10 production in monocytes , and 357 IL-10 has also been demonstrated to suppress LPS-induced IL-6 and IL-12 production We considered the possibility that enhanced production of TNF-a by p i 10a deficient cells might have led to augmented IL-10 production through a paracrine effect and that this may have led suppression of IL-6 and IL-12. Our data indicate that suppression of IL-12 and IL-6 was in fact not explained on this basis (Fig. 5-5 and 5-6). These experiments did not, however, rule out potential paracrine effects from other cytokines not assessed in this study. Interestingly, anti-TNF-a antibodies did moderately suppress IL-6 production in p i 10a deficient cells compared .to isotype matched controls (Fig. 5-5D and 5-6B) and this suggested that TNF-a maybe be required for optimal IL-6 production to some extent. Since we did not examine the effects of these antibodies on . shRNA control cells, we cannot not conclude that TNF-a is similarly required for the control levels of IL-6 production. The independent effects of silencing p i 10a PI3K on the several cytokine studies suggest that this class IA PI3K cannot be classified either as strictly pro-inflammatory or anti-inflammatory in regulating LPS-induced cytokine production. What appears clear, however, is that PI3K p i 10a is required for IL-12 and IL-6 production, while it restricts to limit the production of both TNF-a and IL-10. IL-10 is a potent anti-inflammatory and immunosuppressive cytokine, with effects on T cells and mononuclear phagocytes [reviewed in Ref. 3 8 5 ] . IL-10 regulation is controlled 164 at both the level of transcription and translation " . mRNA-destabilizing motifs like AU-rich' elements (ARE) have been identified in the 3'- untranslated region (UTR) of murine IL-10 mRNA 3 8 S . It has been suggested that many cell types constitutively transcribe IL-10 mRNA and much of the regulation is determined post-transcriptionally 385,388 jj>g j n c i u c e s IL-10 in macrophages, and this requires p38 M A P K and the transcription factor Spl in human monocytes 3 8 9 . The results reported in this thesis show that IL-10 cytokine secretion was significantly enhanced in p i 10a deficient THP-1 cells particularly at later time points. This effect did not appear to be explained by corresponding changes in levels of mRNA (Fig. 5 - 8 ) . These observations suggest that p i 10a may regulate IL-10 production through a negative effect on the translation of IL-10 mRNA, although this was not directly examined. It is also possible p i 10a PI3K may be acting post-translationally. Our finding of.enhanced phosphorylation of p38 M A P K in p i 10a deficient cells (Fig. 5 - 7 B ) was also consistent with increased IL-10 secretion, since IL-10 production is known to be p38 M A P K -dependent 3 8 9 . As discussed above, like TNF-a, IL-10 mRNA also has 3' A R E , however, translational control of IL-10 has not been shown to be dependent on the same mechanism of regulation described for TNF-a by p38 M A P K / M K 2 3 2 6 . This does not, however, rule out the possibility of other mechanisms of translational or post-translational regulation of IL-10 under the control of PI3K. In summary, the results presented in this chapter demonstrate that a specific PI3K isoform can regulate multiple cytokines in response to LPS, with effects that are 165 cytokine-specific. Since both pro-inflammatory and anti-inflammatory signaling pathways have been reported to operate downstream of PI3K [reviewed in Ref. 3 9 0 ] , we propose that both of these effects may be regulated by p i 10a isoform. P O K subunits can physically bind to both key adaptors of TLR4 complex. Thus, p i 10a is able to bind TRIF 3 4 1 , and the p85 subunit can bind to MyD88 3 3 9 . These interactions support a model in which P O K can regulate signaling via both TRIF-dependent and MyD88-dependent pathways. As one example, IRF5 has been shown to regulate LPS-induced IL-6 production downstream of MyD8 8 3 2 7 and endothelial cells expressing DNp85 had 224 defective LPS-induced IL-6 production . Taken together, these findings suggest the possibility that positive regulation of IRF5 by P O K pi 10a downstream of TLR4 could account for our observations of decreased LPS-induced IL-6 production in THP-1 cells where p i 10a has been silenced. P O K may regulate IRF5 by a mechanism similar to that described for TLR3 and IRF3 (Fig. 5-2). In H E K 293T cells expressing TLR3, it was demonstrated that P O K p i 10a activated IRF3 downstream of TLR3 by a specific serine phosphorylation of the IRF3 dimer, leading to transcription of IFN-(3 3 4 2 . Dominant negative Akt abrogated this, leading to the conclusion that Akt was responsible for direct serine phosphorylation of the IRF3 dimer (Fig. 5-2) 3 4 2 . This phosphorylation of IRF3 combined with a second phosphorylation event mediated by T B K l / I K K s , converted IRF3 into an active conformation capable of recruiting higher levels of CBP and binding ISRE ' . Thus p85/pll0a P O K might regulate IRF5 in a similar fashion, to account for the data reported in this thesis. Thus, we propose a model in which activation of TLR4 leads to 166 the recruitment of p85/pll0a PI3K to MyD88 via phosphotyrosine residues, following which pi 10a' brings about phosphorylation of IRF5, possibly through Akt. IRF5 subsequently binds to the ISRE and transcriptional coactivators more efficiently leading to the transcription of proinflammatory cytokines. Further investigations are required to determine whether PI3K pi 10a modulates IRF5 activation in this manner. A model incorporating the present findings on the role of PI3K p i 10a in LPS-induced cytokine expression in human monocytic cells is shown on Figure 5-11. We propose that pi 10a may activate IRF5, similar.to the mechanism described for TLR3/IRF-3 signaling in HEK293 cells in which IRF3 is activated through phosphorylation by the PI3K effector Akt 3 4 2 . The overall effect of IRF5 activation is the induction of proinflammatory cytokines such IL-12p40 and IL-6. This would be consistent with diminished IL-12p40 and IL-6 cytokine mRNA levels seen in LPS-stimulated pi 10a 197 deficient THP-1 cells. So far it is not known what activates IRF5 , but our findings suggest one potential candidate may be PI3K p i 10a. TNF-a may also be positively induced by IRF5, but its transcription in THP-1 cells may be less dependent on IRF5 than on N F K B , as suggested by the fact that IRF5"7" mice were still able to secrete TNF-a 197 albeit at a reduced level . In this model we. propose, therefore, despite reduced IRF5 activation, increased N F K B and p38 phosphorylation in LPS-stimulated p i 10a deficient cells may overcompensate and lead to enhanced TNF-a production. Other potential mechanisms may.explain the decreased responses to LPS by p i 10a deficient cells for IL-6 and IL-12 secretion in the face of increased TNF-a production. 167 PI3K may limit LPS-induced TNF-a production at the transcriptional level, post-transcriptional, or both. In the MyD88-dependent pathway, PI3K may activate PKC^, which may then bring about rapid degradation of IRAK 1 8 6 . This would dampen the 168 Figure 5-11 PI3K p110a. is both a positive and negative regulator in MyD88-dependent pathway Figure 5-11. Model of PI3K in LPS-induced monocyte signaling. L P S and L B P bind to C D 14 and presents it to T L R 4 . T L R 4 / M D 2 undergoes dimerization and activation, 169 which promotes the tyrosine phosphorylation of MyD88 by an unknown kinase. This then leads to the recruitment of p85-pi 10a. TRIF has also been reported to be able to bind p i 10a directly, and recruitment increases with LPS stimulation 3 4 1 . IRF5 can bind directly to TRAF6 and MyD88, and stimulate TNF-a, IL-12p40 and IL-6 through binding to ISRE . These effects are independent of p38 or INK phosphorylation status. On the other hand, IRF4 can mediate negative regulation of cytokine production via direct binding to MyD88 thereby competing with IRF5 binding . In this model pi 10a via its effectors stimulate IRF5 such that downstream IL-12 and IL-6 transcription can occur more efficiently. TNF-a is also activated via N F K B , independent of IRF5, since IRF5"7" mice still can secrete TNF-a albeit at a much lower level 3 2 7 . PI3K p i 10a can limit, but not inhibit TNF-a production. Activation of Akt by PI3K/PDK1 will lead to lower p38 M A P K phosphorylation through inhibition of M A P K K K like ASK-1 or MEKK-3 2 5 5 , 3 9 2 . This leads to less phosphorylation of A R E binding proteins by M K 2 , such that TNF-a mRNA is more transcriptionally repressed. TAK1 maybe a target of PI3K effectors, and inhibition of TAK1 will dampen N F K B activation and p38/JNK phosphorylation. The reduced p38 also limits IL-10 transcription. IRF3 activation for ISFG56 transcription requires PI3K p i 10a in TLR3 signaling 3 4 2 , but its role in TLR4 signaling is not certain. Dashed lines indicate how ECSIT interacts specifically with TRAF6 and M E K K - 1 (another M A P K K K ) and appears to function in this pathway by facilitating the processing of M E K K - 1 3 1 8 . JIP3 (JNK-interacting protein 3) is a TLR4 associated protein that acts as a scaffold protein for JNK 3 9 3 , and this is illustrated to show how a M A P K localizes relative to the TLR4 complex. Arrows indicate activation 170 and blunt arrows indicate inhibition. N F K B activation downstream of RIP-1 is not illustrated. See text for more details. Adapted from Ref. >4>3i8,338,339,34i,342,390,394 171 signal through IRAK/TRAF6/ IKK pathway, and reduce N F K B activation and TNF-a transcription. In IRAKI K O mice, however, TNF-a production in response to LPS was diminished only at low concentrations of LPS (<5 ng/ml) , and in our experiments mRNA levels in LPS-stimulated p i 10a silenced cells were not significantly higher than in control cells (Fig. 5-8). These findings would suggest that the ability of PI3K to limit TNF-a production is not likely mediated solely through IRAK degradation. A more dominant pathway for limiting TNF-a might involve Akt phosphorylation and inhibition of a M A P K K K such as ASK-1 (apoptosis signal-regulating kinase 1) or M E K K - 3 (MAPK/ERK kinase kinase 3) ' . This could lead to restricted activation of p38, less M K 2 activity and correspondingly diminished phosphorylation of A R E binding proteins. As a result these proteins would retain their ability to inhibit translation. Limited activation of p38 M A P K could also restrict IL-10 production to some extent, by 389 restraining activation of Spl . In the model described above and shown schematically in Figure 5-11, PI3K p i 10a through Akt serves to limit overproduction of the early pro-inflammatory cytokine TNF-a, while providing signals to maintain the inflammatory response such as IL-12 that are needed to drive the immune response towards Th l . Interestingly, phospho-JNK and phospho-ERK were not significantly elevated in LPS-stimulated p i 10a deficient cells compared to control cells, suggesting that p i 10a may selectively regulate p38 M A P K . How this may come about remains to be determined. PI3K effectors like Akt may inhibit TAK1 leading to both reduced activation of p38 and N F K B , since human monocytic cells expressing kinase dead TAK1 showed reduced IKK(3 activity in response to LPS 172 treatment, as well as a reduction in p38 and JNK phosphorylation, but not that of E R K It is possible that in our system the effect of p i 10a silencing on JNK was below the level of detection. As shown in Figures 5-2 and 5-11 and discussed elsewhere in this chapter, inhibition of PI3K activity has been shown to bring about enhanced IFNp gene expression in response to LPS via the TRIF-dependent pathway 3 4 ' . In addition, PI3K inhibitors have been demonstrated to enhance IKKa/p phosphorylation, iKB - a degradation and N F K B nuclear translocation in response to LPS in a model of TRIF-mediated N F K B activation 1 9 1 , 3 4 1 . Based upon these findings, we propose a model in which p i 10a exerts negative regulation upstream of the I K K complex in a TRIF-dependent pathway. Perhaps through inhibition of T A K 1 , PI3K may negatively regulate the late phase of N F K B activation as well, which then limits IFN-P production because efficient transcription of IFN-P requires N F K B activation in addition to IRF3 3 4 1 . It remains to be determined whether IRF3 activation in TLR4 signaling is modulated by PI3K, as in the case for TLR3 signaling 3 4 2 , 3 9 1 . Since we did not examine the status of either IRF3 or IFNP in our experiments, we cannot determine i f any of the effects on cytokines by silencing p i 10a is mediated through the TRIF-dependent pathway. Further research will be required to clarify this. The model shown in Figure 5-11 provides a number of working hypotheses for future investigations. Although many of the potential mechanisms illustrated are not established yet, the model highlights the complexity of the roles of PI3K in regulating diverse 173 responses downstream of TLR4. Our data show that p i 10a can regulate several important cytokines independent of other PI3K isoforms. This observation may have been masked by other approaches such as those that used global PI3K inhibitors or mice with targeted disruptions of P O K genes. The inconsistent data from various studies may also reflect cell-type specific effects, in addition to the use of different methodologies and experimental conditions. Many genes are differentially controlled by transcription factors and this depends on the type and differentiation status of the cells Augmentation of IL-10 and inhibition of IL-6 and IL-12 in p i 10a deficient monocytic cells suggests that p i 10a, its effectors, or both may be potential targets for modulating the inflammatory response secondary to LPS exposure or TLR4 activation. Future research focusing on the mechanisms of selective, monocyte cell regulation by pi 10a and other isoforms should reveal candidates for therapeutic targeting. P O K catalytic isoform specific inhibitors have recently been described . These new second generation inhibitors may be useful tools to examine further the role of specific P O K isoforms in animal model of sepsis. In addition, selective silencing of other P O K isoforms by lentiviral delivered siRNA should be informative in respect to assigning specific functions to distinct P O K isoforms. The NADPH-dependent oxidase is important in host defense, and is also involved in the pathogenesis of inflammatory disorders such as arthritis 3 9 8 , ischemic/reperfusion injury 3 9 9 , and others. Understanding the mechanism and regulation of N A D P H oxidase activation is of medical interest because of the potential to help advance therapeutics for 174 modulating the inflammatory response. Experimentally, serum opsonized zymosan is known to fix complement and to bind to CR3 leading to the induction of superoxide production by macrophages and neutrophils 175>243>378>382>400. studies from class IB PI3K pllOy knockout mice have suggested that fMLP-induced superoxide production is regulated by pllOy 1 7 5> 2 3 6. While OPZ-induced oxidase activation was normal in neutrophils from these pllOy knockout mice 1 7 5 , experiments using PI3K inhibitors 378 showed that the oxidative burst to OPZ-zymosan nevertheless required PI3K activity Using THP-1 cells deficient in the class IA PI3K p i 10a isoform, we have now shown that this isoform is required for the OPZ-induced oxidative burst in monocytic cells (Fig. 5-10B). This finding suggests that N A D P H oxidase activation induced by a particulate agonist is brought about by a distinct PI3K isoform from that induced by agonists that stimulate GPCRs, such as fMLP. In our system, unopsonized zymosan did not induce significant superoxide production (Fig. 5-10C). Non-opsonic recognition of zymosan by CR3 is mediated through the lectin domain of CR3, which is distinct from the I/A domain essential for binding and phagocytosis of iC3b-coated particles 4 0 0 . It has been shown that binding zymosan and opsonized zymosan results in differential signaling and mechanisms of phagocytosis 3 6 8 . La Cabec et al showed that uptake of zymosan, in contrast to that of OPZ depends on Rac and cdc42, but not Rho activity . Furthermore, transmission electron microscopy showed that zymosan uptake resembled FcyR-mediated phagocytosis with the appearance of filopodia formation around the particles, while OPZ uptake displayed the classical sinking appearance characteristic of iC3b-coated particle phagocytosis . Lastly, it was 175 found that CR3-mediated phagocytosis of unopsonized zymosan depended on Hck tyrosine kinase, while internalization of OPZ did not . Taken together, these observations suggest that depending on the binding site involved, CR3 can take on different conformations leading to activation of distinct signaling pathways. This may explain why non-opsonized zymosan was unable to induce significant superoxide production in PMA-differentiated THP-1 cells (Fig. 5-10C). However, unopsonized zymosan has been reported to induce superoxide production in other contexts, such as in retinoic acid/vitamin D 3 differentiated U-937 cells 4 0 ° . In this system, OPZ-induced superoxide production was about 1.5 fold over that induced by zymosan. It is possible that use of a distinct differentiation agent or cell type in our experiments may account of these differences. Phorbol esters such as P M A mimic the action of the second messenger diacylglycerol (DAG) and can activate both P K C (classical and novel isoforms) and non-PKC phorbol ester/DAG receptors 4 0 1 , 4 0 2 . In this thesis, high dose P M A induced a strong oxidative burst in control cells, while cells deficient in pi 10a were effectively non-responders (Fig. 5-10A). These findings indicate that—contradictory to the traditional view that PI3K acts upstream of P K C — p i 10a acts downstream of P K C in triggering the oxidative burst. This observation was not entirely unexpected since PI3K products are known to be required to recruit the cytosolic components p47 p h o x and p40 p h o x to the membrane. Studies of ^rara-resveratrol (t-RVT)—a naturally occurring polyphenolic compound found in grapes—are consistent with a model in which p i 10a acts downstream of PKC in activating the oxidase and provide support for this finding 2 3 5 . It has been reported that t-176 RVT inhibited the PMA-induced oxidative burst in human monocytes and an in vitro kinase screen revealed that t-RVT inhibited PI3K by more than 40%, while having no effect on either PDK1 or Akt 2 3 5 . fMLP-stimulated U-937 cells also had increased PI3K activity in phosphotyrosine immunoprecipitates, implicating class IA PI3K activation, and this activity was inhibited by over 70% with t-RVT 2 3 5 . The ability of t-RVT to negatively modulate class IA PI3K activity coupled with its ability to inhibit P M A -induced oxidase activation 2 3 5 again suggests that the latter requires class IA PI3K activity. These findings are consistent with our data showing that p i 10a deficient THP-1 cells were unable to activate the oxidase in response to P M A treatment (Fig. 5-10A), and reveal a novel role for p85/pl 10a PI3K downstream of PKC. It is interesting to consider these results in the context of a recent report that focused on hepatic growth factor signaling. Here it was shown that conventional PKCa acted as a negative regulator of class IA PI3K lipid kinase activity through phosphorylation of the p i 10a subunit 4 0 3 . Whether PKC activates or inactivates PI3K in monocytic cells or can do both, depending upon the context, remains to be determined. Nevertheless, our findings provide clear evidence that PKC is upstream of PI3K in a pathway leading to activation of the monocyte oxidase. It is interesting to compare and contrast this finding with other reported actions of P M A and P K C such as the induction of adherence in monocytic cells independent of PI3K 1 9 2 ' 2 4 4 . While we believe the results presented in Figure 5-1 OA are most likely explained by p85/pll0a acting downstream of PKC, alternative models could be proposed. For example, a,yet unidentified non-PKC phorbol ester/DAG receptor may be involved 177 upstream of P O K p i 10a, leading to PMA-induced oxidase activation. Several non-PKC phorbol ester/DAG receptors have thus far been identified such as: Ras-GRP (guanyl nucleotide-releasing proteins that act as GEFs for Ras and Rapl), Muncl3 isoforms (a family of proteins involved in neurotransmitter release/exocytosis), and chimaerins (GTPase-activating proteins for Rac GTPase) 4 0 ' . The latter are particularly interesting candidates since Rac is a component of the N A D P H oxidase 3 6 0 . It is not clear if any of these proteins or other yet unidentified non-PKC phorbol ester/DAG receptors mediate PMA-induced oxidative burst through P O K (Fig. 5-10A). Further studies will be required to elucidate the mechanism of p85/pl 10a POK-dependent oxidase activation by PMA. The results of our studies focusing on activation of the oxidase are particularly informative when viewed in the context of results obtained from class IB P O K pllOy"7" neutrophils 1 7 5 . Neutrophils from P O K pllOy knockout mice had normal P M A - and OPZ-induced oxidative burst responses, but LPS-primed neutrophils from these mice had defective fMLP-induced burst activity when compared to control cells 1 7 5 . Although similar experiments in macrophages from these mice were not reported, these findings together with our data suggests that class IA P O K p i 10a isoform is required for oxidase activation downstream of CR3 and P M A receptors, while class IB P O K p i lOy is required for the GPCR stimulated oxidative burst. Therefore regulation of the oxidase by P O K provides an example where distinct P O K isoforms can be coupled to distinct receptors to mediate a common biological function. The reasons for this segregation of isoform usage is not entirely clear, but the fact that pi 10a can also mediate other aspects of monocyte 178 biology such as cytokine signaling and phagocytosis suggest that distinct isoform usage allows different cellular processes to be coupled, together depending on the physiological context. 179 C H A P T E R V I : D I S C U S S I O N The signaling pathways in which PI3K family members are involved are complex. This is due to several reasons including: the existence of multiple isoforms with different regulation and enzymatic activities, diverse actions of multiple effectors, and the different tissue distribution and intracellular localization, among the different isoforms. Furthermore, PI3K family members are linked to numerous receptors as well as to diverse regulatory subunits and this also contributes to the challenge of studying their roles in regulating biological processes. This thesis describes a novel approach to silencing PI3K isoforms in human monocytic cells and also identifies several phenotypes brought about by this manipulation. Thus, we show that a deficiency of class IA PI3K p i 10a isoform results in defects involving several important mononuclear phagocyte functions including: adherence, expression of cell surface receptors, phagocytosis, activation of the phagocyte oxidase, and cytokine secretion in response to bacterial endotoxin. The data show that in respect to these properties p85/pl 10a PI3K appears to have both redundant and non-redundant roles. Several generalizations of this isoform specificity as well as the limitations of the gene-silencing approach used are discussed below. 6.1 Choice of gene silencing strategy may affect phenotype. Different PI3K gene silencing strategies can result in different signaling and functional outcomes 6 0 ' 4 0 4 . The non-specific or bystander effects of alterations in PI3K subunit expression seen in some PI3K knockout experiments were discussed in Chapter III. We 180 show here that—in contrast to the non-specific effects observed with the generation of p85a knock-out mice ' —lentiviral-delivered siRNA provides a means to achieve specific silencing of a PI3K family member without altering the expression of other isoforms. The particular gene targeting strategy used may also affect experimental outcomes by virtue of the occurrence of PI3K kinase-independent effects. Many functions attributed to PI3K signaling have been discovered through the use of PI3K inhibitors, which inhibit the kinase function of the catalytic p i 10 subunit. The p i 10 subunit, however, has multiple domains that can mediate non-kinase dependent effects, and therefore gene knockouts that remove the entire catalytic subunit will not be able to distinguish between kinase and non-kinase dependent functions 4 0 4 . A good example illustrating this is the effect on cardiac phenotype by class IB PI3K pllOy D 0 . PI3K pllOy K O mice had, in addition to its immunological phenotypes (Table 1-4), increased cardiac contractility and (TO developed cardiac tissue damage under pressure overload . One mechanism proposed to suggest how p i lOy regulates contractility is by reducing basal levels of cAMP 4 0 5 . In experiments by Hirsch and colleagues, mice with a complete knockout of PI3K pllOy isoform were compared with mice expressing a kinase-dead p i lOy, created by "knocking in" an inactivating point mutation of the catalytic domain. They discovered that pllOy kinase-dead mice did not develop myocardial damage and did not have elevated cAMP concentrations 6 8 . Kinase dead P O K pllOy was shown to bind and activate phosphodiesterase 3B (PDE3B), which mediated cAMP hydrolysis. Therefore, the cardiac P O K pllOy is able to participate in a kinase-independent activity that relies on 181 protein interactions to regulate PDE3B activity and negatively modulate cardiac contractility. This example illustrates the importance of protein-protein interactions and scaffolding in the control of signaling pathways 4 0 4 , and highlights the limitation of gene silencing strategies. This kinase-independent effect of pllOy isoforms have not been identified in class IA PI3K 4 0 6 , and was not explored using the lentiviral approach presented in this thesis. However, we cannot exclude the possibility that kinase-independent functions of p i 1 Oa isoform may be partly responsible for the phenotypes observed. A recent report of newly developed PI3K class I isoform specific inhibitors 3 1 ' 4 0 7 may be useful tools in clarifying this issue by doing studies using a combination of specific enzymatic inhibitors with approaches that down-regulate protein levels. Another limitation of the results presented in this thesis is that experiments did not address changes in 3'-PI levels or location in the cells brought about as a result of silencing p i 10a by lentiviral delivered siRNA. Overall levels of PtdIns(3,4,5)P3 in the cell after stimulation could be normal even in p i 10a silenced cells since other class I isoforms are still active, and may be activated through other pathways. Therefore, phenotypic features attributed to p i 10a may be due to alterations in subcellular localization of PtdIns(3,4,5)P3 or other metabolites rather than to overall reductions in their levels. Studies using GFP tagged probes with PH or P X domains might help to localize changes in the subcellular distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and allow further insight into mechanisms. 182 6.2 A single receptor complex can activate multiple PI3K isoforms, and different receptors can lead to the same biologic response by using different PI3K isoforms Results from Chapter 4 on LPS and D3-induced adherence illustrate how different PI3K isoforms can lead to a common function through associations with different receptors (Fig. 6-1A). Our data show that p i 10a was required for D 3 but not LPS-induced adherence, even though the latter was also a PI3K dependent process (Fig. 4-1). The PI3K isoform mediating LPS-induced adherence may be a distinct p i 10 enzymatic subunit or it may belong to another class of PI3K. Similar results to these have been observed in other systems. For example in mast cells, kit-induced exocytosis of secretory granules and Akt phosphorylation used class IA PI3K, while FcsRI-initiated exocytosis and Akt phosphorylation was dependent on PI3K activity, but independent of class IA 1 89 PI3K . These findings suggest a common theme of receptor-specific usage of different PI3K isoforms to regulate identical cellular functions. Future experiments using siRNA targeting of other PI3K isoforms will help to determine which isoform mediates LPS-induced adherence. It is interesting and informative to contrast these results with our studies of cytokine production in response to LPS. We found that unlike the case with LPS-induced adherence, p i 10a was required for LPS-induced cytokine production (Chapter 5). Taken together, these findings suggest that a given receptor/receptor complex can use multiple PI3K isoforms to bring about diverse biological responses to the same agonist. The factors that govern isoform recruitment by any specific receptor are likely to be complex 183 and are a focus of particular interest. For example it was recently shown that pllOp but not pi 10a, was able to bind the early endosomal marker Rab5 through amino acid residues that are specific to p i 10(3 4 0 8 . This suggests that under both basal and stimulated conditions, various class IA PI3K isoforms may have different subcellular localizations influenced at least in part by their sequence differences. These differences may partially account for the mechanism of differential isoform recruitment by receptor complexes and even for recruitment of distinct isoforms to separate regions of the same receptor. 6.3 Control of diverse monocyte effector functions by a single isoform of PI3K Phagocytosis and the subsequent killing of microbes in the phagosome form the basis of innate immunity against pathogens . Assembly of an activated N A D P H oxidase on the phagosomal membrane.is crucial for the microbicidal function of phagocytes and host defense against infections as has been made evident by patients with chronic granulomatous disease 2 2 9 . Findings reported in Chapters 4 and 5 show that both phagocytosis and activation of the N A D P H oxidase is dependent p85/pl 10a PI3K. This suggests that common isoform usage may allow different cellular processes to be coupled together (Fig. 6 - 1 B ) . Depending on the context, however, the pathways and functions described here are not exclusive to p85/pl 10a PI3K. For example, class IB p i 10y PI3K was shown to regulate the oxidative burst downstream of fMLP receptors 1 7 5 , and phagocytosis of apoptotic cells required pllOp in murine macrophages 2 2 2 . Although in the latter study N A D P H oxidase activation was not examined, it has been reported that phagocytosis of apoptotic Jurkat cells by murine macrophages had attenuated P M A -184 induced oxidative burst 4 0 9 . Taken together, these findings suggest that inflammatory responses may or may not be coupled to phagocytosis. Furthermore, when coupling occurs it may be mediated by p85/pll0a PI3K downstream of specific phagocytic receptors, as in the case of FcyR- or CR3-rriediated phagocytosis. In addition, other receptors that activate PI3K like TLR also contribute to proinflammatory signals. Further research will be required to test this hypothesis. 185 Figure 6-1 Figure 6-1. Model of PI3K isoform usage linked to monocyte receptors and functional responses. A, Different receptors use distinct PI3K isoforms to regulate a common cellular function, and a single receptor complex can activate multiple PI3K isoforms. Abbreviations: L Y , LY294002. Wm, wortmannin. B, A single PI3K isoform regulates multiple monocyte effector functions. In addition to adherence and cytokine production (panel A ) PI3K p i 10a isoform is required for efficient phagocytosis through FcyR. This may be mediated through myosin X and A R N O for respectively pseudopod extension and focal endomembrane insertion. It is not clear i f myosin X is also required for CR3-mediated phagocytosis. The same PI3K isoform is then able to activate the N A D P H oxidase, an important component in the microbicidal activity against ingested microbes. 1 8 6 6.4 Perspectives and significance Mononuclear phagocytes play crucial roles in both the innate and acquired immune responses. The 3'-PI produced by PI3K regulate many processes in monocytic cells 47,6o,i86,i87,i89,244,4io I n a d d i t i o r l j i m p a i r e d r e g u l a t i o n of PI3K pathways in immune cells has been demonstrated in several immunological processes and diseases 4 0 6 . Knockouts of pllOy and p i 105 in mice have revealed defective recruitment of neutrophils and macrophages to inflammatory sites 1 7 5 , and indicated that these PI3K isoforms are involved in inflammatory bowel disease 4 1 a n d autoimmune renal disease 4 1 2 (Table 1-4 ) . Because gene targeting of class IA p i 10a and p i 10(3 was found to be lethal in mice 6 0 , less has been known about their contribution to both normal and pathological conditions. Using lentiviral-delivered siRNA, we were able to identify important roles for p i 10a in the regulation of adherence, phagocytosis, the phagocyte oxidase, and cytokine production in human mononuclear phagocytes. The fine specificity of how these critical effector functions are regulated is a major focus for future investigations. Elucidation of the precise contribution of PI3K isoforms could lead to the development of isoform specific approaches to promote a selective therapeutic action while minimizing deleterious side effects 6 7>3 9 7. A clear understanding of the contribution of PI3K enzymes to monocyte cell regulation— through the use of siRNA—has important implications beyond the immunological sciences. For example monocytes and macrophages are critical to the.pathogenesis of atherosclerosis 6 , 4 1 3 and lentiviral based strategies for gene silencing of PI3K should provide important insights here. Furthermore, PI3K has been implicated in a variety of 187 non-immune diseases such as cardiac failure 6 8>4 0 5 5 type II diabetes 4 1 4 , hypertension 4 1 5 , 4 1 6 3 and thromboembolism 4 1 7 where mononuclear phagocytes are found at sites of tissue damage. Therefore, POKs are promising targets for therapeutic intervention 6 0 , 3 9 7 . PI3K is known to promote diverse cellular properties such as proliferation, anti-apoptosis, and migration 6 7 which can contribute to oncogenesis. Thus, it is not surprising that heterogeneous defects in the PI3K-Akt signaling pathway have been identified with a high frequency in human tumors 4 1 8 . In fact, somatic mutations in the PIK3CA gene, which encodes p i 10a isoform, has been implicated in a variety of tumors [reviewed in Ref. 4 I 9 ] . For example, studies have shown a PIK3CA mutation in up to 32% of colorectal cancers 4 2 0 , 5-27% of glioblastomas 4 2 0 ' 4 2 1 ; 6.5-25% of gastric cancers 4 2 0 - 4 2 2 ; 36% of hepatocellular carcinomas 4 2 2 , 8-40% of breast cancers 4 2 0 ' 4 2 2 - 4 2 4 , 4.12% of ovarian cancers 4 2 3 , and 1.3-4% of lung cancers 4 2 0 - 4 2 2 . 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