Disruption of Androgen Metabolism, Regulation and Effects: Involvement of Steroidogenic Enzymes Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Cornelia Fürstenberger, aus Basel, Schweiz Basel, 2014 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Alex Odermatt (Fakultätsverantwortlicher) ________________________ Fakultätsverantwortlicher Prof. Dr. Alex Odermatt und Prof. Dr. Rik Eggen (Korreferent) ________________________ Korreferent Prof. Dr. Rik Eggen Basel, den 20. Mai 2014 ________________________ Dekan Prof. Dr. Jörg Schibler 2 Table of content Table of content I. Abbreviations ................................................................................................................................. 6 1. Summary ........................................................................................................................................ 9 2. Introduction .................................................................................................................................. 12 2.1 Steroid hormones .................................................................................................................. 12 2.2 Steroidogenesis ..................................................................................................................... 14 2.3 Steroid hormones in health and disease ............................................................................... 15 2.4 Hydroxysteroid dehydrogenases are key pre-receptor control elements regulating the balance between active and inactive hormones ................................................................................ 15 2.5 11β-Hydroxysteroid dehydrogenase type 1 and type 2 ......................................................... 17 2.5.1 Properties and expression of 11β-hydroxysteroid dehydrogenases ............................. 17 2.5.2 11β-Hydroxysteroid dehydrogenase type 2 is co-expressed with the mineralocorticoid receptor in epithelial cells .............................................................................................................. 18 2.5.3 11β-Hydroxysteroid dehydrogenases 1 interacts with hexose-6 phosphate dehydrogenase .............................................................................................................................. 18 2.5.4 11β-Hydroxysteroid dehydrogenases in health and disease ........................................ 18 2.6 Focus on the short chain dehydrogenase/reductase family member 17β-hydroxysteroid dehydrogenase type 3 ....................................................................................................................... 20 2.6.1 Properties and expression of 17β-hydroxysteroid dehydrogenase type 3 .................... 20 2.6.2 17β-Hydroxysteroid dehydrogenase type 3 deficiency ................................................. 21 2.7 Focus on the aldo-keto reductase family member 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3) .......................................................................................................................................... 22 2.7.1 The role of 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3) in prostate cancer .. 22 2.8 The pleiotropic effects of testosterone .................................................................................. 23 2.8.1 Male embryonic development is testosterone dependent ............................................. 23 2.8.2 Male fertility and sexual health is driven by testosterone .............................................. 23 2.8.3 The role of testosterone in metabolism ......................................................................... 23 2.8.4 Testosterone and prostate cancer - a paradigm shift .................................................... 24 2.8.5 The role of testosterone in male breast cancer is not yet understood .......................... 25 2.8.6 Testosterone and cognition ........................................................................................... 25 2.8.7 The Janus face of testosterone when it comes to muscles, bones and strengths - clinic and doping ..................................................................................................................................... 26 3. Project 1: Evaluation of anabolic androgenic steroids regarding their effect on 11β- hydroxysteroid dehydrogenase 2-dependent glucocorticoid inactivation ................................... 27 3.1 Introduction anabolic androgenic steroids ............................................................................. 27 3.1.1 The different types of ASS users - prevalence data ...................................................... 27 3.1.2 Abuse of AAS - what are the consequences? ............................................................... 28 3.2 Are AAS inhibitors of 11β-hydroxysteroid dehydrogenase 2 and may therefore cause cortisol-induced mineralocorticoid receptor activation? .................................................................... 29 3 Table of content 3.3 Published Paper: The anabolic androgenic steroid fluoxymesterone inhibits 11β- hydroxysteroid dehydrogenase 2–dependent glucocorticoid inactivation ......................................... 30 3.4 Further results and outlook concerning 11β-hydroxysteroid dehydrogenase type 2 inhibition by androgenic steroids ...................................................................................................................... 40 3.4.1 Androstenedione potently inhibits 11β-hydroxysteroid dehydrogenase type 2 ............. 40 4. Project 2: Evaluation of testosterone production in Leydig cell models and characterization of current methods by measuring androgen levels ............................................ 42 4.1 Introduction to endogenous androgens ................................................................................. 42 4.2 Technical methods of steroid measurements ........................................................................ 43 4.2.1 Immunoassays ............................................................................................................... 44 4.2.2 Mass spectrometry ........................................................................................................ 44 4.3 Overview of the testis and testicular cell types ...................................................................... 45 4.4 Pathways involved in androgen formation – front-door versus back-door ............................ 46 4.5 Leydig cell models in endocrine research ............................................................................. 48 4.6 Aims of the Leydig cell characterization project .................................................................... 49 4.7 Paper Draft ............................................................................................................................ 50 Comparison of androgen synthesis in MA-10 and BLTK-1 mouse Leydig cells ............................... 50 4.7.1 Abstract .......................................................................................................................... 50 4.7.2 Introduction .................................................................................................................... 50 4.7.3 Materials and Methods .................................................................................................. 51 4.7.4 Results ........................................................................................................................... 54 4.7.5 Discussion ..................................................................................................................... 58 5. Project 3: Transcriptional regulation of 17β-hydroxysteroid dehydrogenase ...................... 61 5.1 Introduction ............................................................................................................................ 61 5.1.1 Possible consequences of 17β-hydroxysteroid dehydrogenase type 3 up regulation .. 61 5.1.2 Inflammation, tumor necrosis factor-α and cancer ........................................................ 62 5.2 Our approach: Study the transcriptional regulation of 17β-hydroxysteroid dehydrogenase 3 .. ............................................................................................................................................... 64 5.3 Focus part A: The inflammatory cytokine TNF-α activates the promoter of 17β- hydroxysteroid dehydrogenase 3 ...................................................................................................... 64 5.4 Part A manuscript draft: TNF-α stimulates testosterone production by p38 MAPK-dependent activation of 17β-hydroxysteroid dehydrogenase 3 promoter ........................................................... 65 5.4.1 Abstract .......................................................................................................................... 65 5.4.2 Introduction .................................................................................................................... 65 5.4.3 Materials and Methods .................................................................................................. 67 5.4.4 Results ........................................................................................................................... 70 5.4.5 Discussion ....................................................................................................................