Investigating caveolin-dependent glucocorticoid action in inflammation A thesis submitted to the University of Manchester for the degree of M.Phil in the Faculty of Medicine and Human Sciences 2013 Anna Louise Mary Mannion-Jones School of Medicine 1 Contents I. TABLES AND FIGURES .................................................................................................. 7 II. Abstract .................................................................................................................... 10 III. Declaration ............................................................................................................ 10 IV. Notice of Copyright................................................................................................ 11 1 Introduction .............................................................................................................. 18 1.1 Inflammation ..................................................................................................... 18 1.1.1 The cellular inflammatory response ........................................................... 18 1.1.2 Innate vs. adaptive immunity ..................................................................... 19 1.1.3 Chronic inflammation ................................................................................. 20 1.1.4 Autoimmune disease.................................................................................. 20 1.1.5 Anti-inflammatory actions of glucocorticoids ............................................. 22 1.1.6 The development of synthetic glucocorticoids ............................................ 22 1.1.7 Side effects of glucocorticoid treatment ..................................................... 23 1.2 The glucocorticoid receptor ............................................................................... 24 1.2.1 The structure of the glucocorticoid receptor .............................................. 25 1.2.2 The N-terminal domain of the glucocorticoid receptor ............................... 25 1.2.3 The DNA-binding domain of the glucocorticoid receptor ............................ 25 1.2.4 The ligand binding domain of the glucocorticoid receptor .......................... 25 1.2.5 Post-translational modifications of the glucocorticoid receptor .................. 26 1.2.6 Glucocorticoid receptor isoforms ............................................................... 26 1.2.7 Alternative translation start sites generates glucocorticoid receptor subtypes 27 1.3 Glucocorticoid receptor effects: Genomic signalling ........................................... 30 1.3.1 GR binding to DNA ..................................................................................... 30 1.3.2 GR tethering to other DNA bound transcription factors. ............................. 32 1.4 Glucocorticoid receptor effects: Non-genomic signalling .................................... 34 1.4.1 The stress response .................................................................................... 34 2 1.4.2 Mechanisms of non-genomic signalling ...................................................... 35 1.5 Cross talk with other signalling pathways and the relationship between genomic and non-genomic signalling mechanisms ....................................................................... 40 1.5.1 Post-translational modifications of proteins in signalling: Phosphorylation . 42 1.5.2 Other inhibitors of NF-κB inflammatory signalling ....................................... 43 1.6 Caveolin and caveolae ........................................................................................ 43 1.6.1 Caveolin knockout mice .............................................................................. 46 1.6.2 Caveolin, NF-κB and pulmonary inflammation and fibrosis ......................... 47 1.6.3 Caveolin and inflammatory pathways ......................................................... 49 1.6.4 GR association with caveolin ...................................................................... 50 1.7 Project Outline ................................................................................................... 53 1.7.1 Hypothesis ................................................................................................. 54 1.7.2 Aim ............................................................................................................ 54 1.1.1 Major techniques to be used to investigate aims ........................................ 55 2 Materials and Methods .............................................................................................. 59 2.1 Mammalian cell culture ...................................................................................... 59 2.1.1 Cell maintenance ........................................................................................ 59 2.1.2 Cryoprocessing ........................................................................................... 59 2.1.3 Plating cells for experiments ....................................................................... 59 2.1.4 Treatment with Dexamethasone ................................................................ 60 2.2 Animal housing .................................................................................................. 61 2.2.1 In vivo Experimental design ........................................................................ 61 2.2.2 Animal treatment ....................................................................................... 61 2.3 Plasmid preparation ........................................................................................... 62 2.3.1 Miniprep .................................................................................................... 63 2.3.2 Restriction Digest ....................................................................................... 63 2.3.3 Maxiprep .................................................................................................... 63 2.4 Transfections ..................................................................................................... 64 3 2.4.1 Plasmid transfection................................................................................... 64 2.4.2 Transfection of siRNA against Caveolin ....................................................... 64 2.5 Western blot ...................................................................................................... 64 2.5.1 Extracting protein from Cells ...................................................................... 64 2.5.2 Extracting protein from fresh tissues .......................................................... 65 2.5.3 Extracting protein from snap-frozen tissues ............................................... 66 2.5.4 SDS Gel Electrophoresis and transfer of proteins to membrane .................. 66 2.5.5 Protein labelling with antibodies ................................................................ 67 2.6 Antibodies used for protein detection in Western blot and Immunofluorescence 67 2.7 Real Time Quantitative Polymerase Chain Reaction (RT-QPCR) .......................... 68 2.7.1 RNA extraction from cells ........................................................................... 68 2.7.2 RNA extraction from lung tissue ................................................................. 69 2.7.3 Setting up the Real-Time qPCR reaction ..................................................... 70 2.7.4 Real-Time qPCR reaction Thermal Cycler .................................................... 70 2.7.5 Analysis of QPCR results by ΔΔCT ............................................................... 70 2.7.6 Primer design and validation ...................................................................... 71 2.7.7 Sequences of primers used in QPCR ........................................................... 72 2.8 Genotyping ........................................................................................................ 72 2.9 Live cell microscopy ........................................................................................... 74 2.9.1 Analysis of GR translocation in Live Cell experiment ................................... 74 2.10 Immunofluorescence in Cells ............................................................................. 74 2.10.1 Cell fixing and permeabilization .................................................................. 74 2.10.2 Antibody labelling ...................................................................................... 75 2.10.3 Imaging Immunofluorescence .................................................................... 75 2.10.4 Phalloidin Actin stain with fluorescent FITC dexamethasone ...................... 75 2.11 Immunohistochemistry in tissues ....................................................................... 77 2.11.1 FFPE (formalin fixed paraffin embedded) Tissue processing........................ 77 4 2.11.2 Antibody labelling of tissue sections, Immunofluorescence ........................ 77 2.11.3 Nuclei counterstaining ................................................................................ 78 2.11.4 Quenching of autofluorescence using UV and Sudan Black B ...................... 78 2.11.5 Visualising fluorescent antibody labelling in tissues .................................... 78 2.12 Bronchoalveolar lavage ...................................................................................... 79 2.13 Serum Corticosterone ELISA ..............................................................................