Glucocorticoids recruit Tgfbr3 and Smad1 to shift transforming growth factor-β signaling from the Tgfbr1/Smad2/3 axis to the Acvrl1/Smad1 axis in lung fibroblasts Inauguraldissertation zur Erlangung des Grades eines Doktors der Medizin des Fachbereichs Medizin der Justus-Liebig-Universität Gießen vorgelegt von Schwartze, Julian Tristan aus Langen (Hessen), Deutschland Gießen (2014) Aus dem Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center, German Center for Lung Research, Giessen, Germany. Leiter/Direktor: Prof. Dr. med. Werner Seeger Gutachter: Prof. Dr. med. Werner Seeger Gutachter: Prof. Dr. Klaus T. Preissner Tag der Disputation: 31.07.2015 Table of contents I I. Table of contents I. Table of contents ................................................................................................ I II. List of figures .................................................................................................... VI III. List of tables ................................................................................................... VIII IV. List of abbreviations ......................................................................................... IX V. Summary ........................................................................................................... XI VI. Zusammenfassung .......................................................................................... XII 1. Introduction ............................................................................................................ 1 1.1 Background .......................................................................................................... 1 1.2 Transforming growth factor-β ............................................................................. 1 1.2.1 TGF-β ligands ............................................................................................. 2 1.2.2 Activation of TGF-β ..................................................................................... 2 1.2.3 TGF-β receptors .......................................................................................... 4 1.2.4 Signal transduction mechanism and Smad proteins .................................... 4 1.2.5 Gene regulation ........................................................................................... 6 1.2.6 TGF-β co-receptors ..................................................................................... 7 1.2.7 Betaglycan: an accessory TGF-β receptor .................................................. 7 1.2.8 Tgfbr1 versus Acvrl1 signaling ..................................................................... 8 1.2.9 TGF-β signaling in lung development .......................................................... 9 1.2.10 TGF-β signaling in lung disease ................................................................ 9 1.2.11 Fibroblast-to-myofibroblast differentiation ................................................ 12 1.3 Glucocorticoids .................................................................................................. 13 1.3.1 Glucocorticoid effects in the human body .................................................. 13 1.3.2 Glucocorticoid cell signaling ...................................................................... 13 1.3.3 The intracellular glucocorticoid receptor .................................................... 13 1.3.4 Signal transduction mechanism ................................................................. 14 1.3.5 Regulation of glucocorticoid concentrations in the body ............................ 15 1.3.6 Cortisol synthesis ...................................................................................... 15 1.3.7 Cortisol derivatives .................................................................................... 16 1.3.8 General clinical use of glucocorticoids ....................................................... 16 1.3.9 Adverse effects of glucocorticoids ............................................................. 16 1.3.10 Glucocorticoids in TGF-β-related lung pathologies and development ...... 17 Table of contents II 2. Aim of the study ................................................................................................... 20 3. Materials and methods ......................................................................................... 21 3.1 Materials .............................................................................................................. 21 3.1.1 Equipment ................................................................................................. 21 3.1.2 Reagents ................................................................................................... 22 3.2 Methods .............................................................................................................. 24 3.2.1 Cell culture ................................................................................................ 24 3.2.1.1 NIH/3T3 mouse fibroblast-like cells ..................................................... 24 3.2.1.2 Isolation of primary mouse lung fibroblasts ......................................... 24 3.2.1.3 Cultivating primary mouse lung fibroblasts .......................................... 25 3.2.1.4 Primary human lung fibroblasts ........................................................... 26 3.2.1.5 Primary human pulmonary artery endothelial cells .............................. 26 3.2.1.6 Primary human pulmonary artery smooth muscle cells ....................... 26 3.2.1.7 H441 human Clara cell-like airway epithelial cells ............................... 26 3.2.1.8 Experimental cell culture setup ........................................................... 26 3.2.2 mRNA isolation ......................................................................................... 27 3.2.2.1 mRNA isolation from cells ................................................................... 27 3.2.2.2 mRNA isolation from mouse lung, heart, liver, and kidney................... 27 3.2.3 Determining mRNA concentrations ........................................................... 27 3.2.4 Reverse transcription reaction ................................................................... 27 3.2.5 Real-time polymerase chain reaction ......................................................... 28 3.2.6 Determining mRNA expression by StepOne Software ............................... 29 3.2.7 Protein isolation ......................................................................................... 30 3.2.7.1 Protein isolation from cells .................................................................. 30 3.2.7.2 Protein isolation from mouse lungs ..................................................... 30 3.2.8 Determining protein concentrations ........................................................... 30 3.2.9 SDS polyacrylamide gel electrophoresis ................................................... 31 3.2.10 Immunoblot analysis ................................................................................ 32 3.2.10.1 Immunoblotting ................................................................................. 32 3.2.10.2 Protein visualization .......................................................................... 33 3.2.10.3 Membrane stripping .......................................................................... 34 3.2.10.4 Determining protein density by Multi Gauge MFC Software .............. 34 3.2.11 Transfection of cells with small interfering RNA ....................................... 34 3.2.11.1 Experimental cell culture setup for knock-down experiments ............ 35 3.2.12 Transfection of cells with TGFBR3-expressing plasmid constructs .......... 35 Table of contents III 3.2.12.1 Experimental cell culture setup for overexpression experiments ....... 35 3.2.13 Dual luciferase ratio assay analysis ......................................................... 36 3.2.13.1 Transfection of cells with corresponding plasmids ............................. 36 3.2.13.2 Experimental cell culture setup for dual luciferase assay................... 37 3.2.13.3 Experimental cell culture setup with small interfering RNA ................ 37 3.2.13.4 Experimental cell culture setup with TGFBR3-expressing plasmids .. 38 3.2.13.5 Cell lysis ............................................................................................ 38 3.2.13.6 Determining luciferase activity ........................................................... 38 3.2.14 Animal studies ......................................................................................... 38 3.2.14.1 Pulmonary effects of dexamethasone in mice ................................... 38 3.2.14.2 Organ storage ................................................................................... 39 3.2.15 Statistical analysis ................................................................................... 39 4. Results .................................................................................................................. 40 4.1 Glucocorticoids inhibit classical Tgfbr1/Smad2/3 signaling in NIH/3T3 mouse fibroblast-like cells....................................................................................... 40 4.1.1 p(CAGA)9 induction analysis by dual luciferase assay ............................... 40 4.1.2 Immunoblot analysis