Centre for Cell Signalling Barts & The London School of Medicine and Dentistry Queen Mary University of London THE ROLE OF PI3K p110δ IN THE REGULATION OF GENE EXPRESSION IN MURINE MACROPHAGES PhD Thesis Postgraduate Student: Emily Burns Supervisors: Prof. B. Vanhaesebroeck & Dr. B. Twomey Sponsors: BBRSC and UCB 1 Abstract PI3K are a family of lipid kinase enzymes that are involved in a broad spectrum of cellular, physiological and pathological processes. Notably, PI3K dysfunction is associated with cancer, inflammation and metabolism. The PI3K family consists of three classes, Class I, Class II and Class III, which are defined according to their structure, function and lipid specificity. Class I is further subdivided into Class IA, which consists of p110, p110 and p110, and Class IB, which consists of p110. Both p110 and p110 are ubiquitously expressed. Conversely, p110and p110 have a restricted tissue distribution, limited mainly to leukocytes, where they are highly enriched. In this work, we have focused on understanding the role of p110 in macrophages and specifically, on gaining insight into its role in genome-wide transcriptional regulation. Our results suggest that the contribution of p110δ activity to transcriptional regulation in growing primary macrophages is very limited. Genetic or pharmacological inactivation of p110δ resulted in differential regulation of less than twenty-five unique genes. Interestingly, at the level of genome-wide transcription, we have observed a significant difference between the effects of pharmacological and genetic inactivation. Indeed, none of the genes identified were differentially regulated as a result of both pharmacological and genetic inactivation of p110δ. We discovered that the majority of the genes differentially regulated in p110δ KI macrophages are located within close proximity to the Pik3cd gene. We propose that the differential regulation of these genes represents an artefact of the genetic engineering strategy employed to generate the p110δ KI mice. We have confirmed that genetic inactivation of p110δ results in reduced expression of Rab6b, a gene which is not linked to Pik3cd. We have confirmed that this does translate into a reduced level of rab6b protein in p110δ KI macrophages. Parts of this thesis have been published elsewhere: Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages Evangelia A. Papakonstanti, Olivier Zwaenepoel, Antonio Bilancio, Emily Burns, Gemma E. Nock, Benjamin Houseman, Kevan Shokat, Anne J. Ridley and Bart Vanhaesebroeck, TJournal of Cell Science [1] 2 Contents LIST OF FIGURES ......................................................................................................................................... 8 LIST OF TABLES ........................................................................................................................................ 11 1. INTRODUCTION .............................................................................................................................. 16 1.1. THE PI3K FAMILY OF ENZYMES .................................................................................................................. 16 1.1.1. Class I PI3K ....................................................................................................................... 17 1.2. CELLULAR SIGNALLING BY CLASS IA PI3K ISOFORMS ............................................................................. 18 1.2.1. Activation of Class IA PI3K isoforms ....................................................................... 18 1.2.2. Signalling downstream of Class IA PI3K activation ......................................... 20 1.2.3. Temporal regulation of PI3K signalling ................................................................ 20 1.2.4. Akt ........................................................................................................................................ 21 1.2.5. Regulation of transcription downstream of PI3K-Akt pathway ............................................................................................................................. 24 1.3. INVESTIGATING THE PHYSIOLOGICAL AND PATHOLOGICAL IMPLICATIONS OF PI3K ACTIVITY ............................................................................................................................................... 31 1.3.1. Tools and models for investigating PI3K functions .......................................... 31 1.3.2. Non-redundant cell-specific functions of Class IA PI3K isoforms ................................................................................................................... 35 1.4. CELLULAR AND PHYSIOLOGICAL ROLES OF CLASS IA PI3KS .................................................................. 36 1.4.1. Cellular and physiological roles of p110α ............................................................ 36 1.4.2. Cellular and physiological roles of p110β............................................................. 37 1.4.3. Cellular and physiological roles of p110δ ............................................................. 37 1.4.4. Cellular and physiological roles of p110γ ............................................................. 40 1.5. MACROPHAGES IN THE IMMUNE SYSTEM ................................................................................................... 42 1.5.1. The components of the immune system and stages of immune response ............................................................................................................ 42 1.5.2. Initiation of the adaptive immune response ........................................................ 43 1.5.3. Recognition of lipopolysaccharide and other pathogens by macrophages ....................................................................................... 44 1.5.4. The importance of a balanced immune response .............................................. 50 1.6. THERAPEUTIC POTENTIAL OF PI3K INHIBITION ...................................................................................... 52 1.6.1. Therapeutic potential of targeting p110α ........................................................... 52 1.6.2. Therapeutic potential of targeting p110β ........................................................... 52 1.6.3. Therapeutic potential of targeting p110δ............................................................ 53 1.7. PROJECT AIMS AND OBJECTIVES ...................................................................................................... 58 2. MATERIALS AND METHODS ....................................................................................................... 59 2.1. MICE ................................................................................................................................................................. 59 2.1.1. Genotyping of mice ........................................................................................................ 59 3 2.2. REAGENTS, SOLUTIONS AND BUFFERS ........................................................................................................ 61 2.2.1. Inhibitors ........................................................................................................................... 62 2.2.2. Antibodies .......................................................................................................................... 63 2.3 CELL CULTURE ................................................................................................................................................ 64 2.3.1 Macrophage cell lines ................................................................................................... 64 2.3.2 Isolation and differentiation of primary macrophages .................................................................................................................... 65 2.3.3 Cell stimulation ............................................................................................................... 68 2.4 PROTEIN ANALYSIS AND DETECTION .......................................................................................................... 68 2.4.1 Western blot analysis .................................................................................................... 68 2.4.2 Semi-quantitative analysis of PI3K activation using p-Akt to Akt ratios .......................................................................................................... 71 2.5 FLUORESCENCE ACTIVATION CELL SORTING (FACS) .............................................................................. 71 2.5.1 Preparation and staining of cells ............................................................................. 71 2.5.2 Analysis of stained cell by FACS ................................................................................ 71 2.6 EXTRACTION OF CELLULAR RNA AND QUALITY CONTROL ...................................................................... 72 2.6.1 Cell lysis .............................................................................................................................. 72 2.6.2 RNA extraction ................................................................................................................ 72 2.6.3 RNA quantification and purity assessment .......................................................... 72 2.7 QUANTITATIVE REAL-TIME PCR (QPCR) ................................................................................................. 73 2.7.1 Reverse transcription