ELINA NUMMENMAA TRPA1 as a Novel Factor and Drug Target in Osteoarthritis Tampere University Dissertations 210 Tampere University Dissertations 210 ELINA NUMMENMAA TRPA1 as a Novel Factor and Drug Target in Osteoarthritis ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine and Health Technology of Tampere University, for public discussion in the auditorium F115 of the Arvo building, Arvo Ylpön katu 34, Tampere, on 7 February 2020, at 12 o’clock. ACADEMIC DISSERTATION The Immunopharmacology Research Group, Faculty of Medicine and Health Technology, Tampere University Tampere University Hospital Coxa Hospital for Joint Replacement Finland Responsible Professor Eeva Moilanen supervisor Tampere University and Custos Finland Supervisor Docent Katriina Vuolteenaho Tampere University Finland Pre-examiners Professor Jari Arokoski Ari-Pekka Koivisto, PhD University of Helsinki Stockholm University Finland Sweden Opponent Professor Ali Mobasheri State Research Institute Centre for Innovative Medicine Lithuania The originality of this thesis has been checked using the Turnitin OriginalityCheck service. Copyright ©2020 author Cover design: Roihu Inc. ISBN 978-952-03-1434-7 (print) ISBN 978-952-03-1435-4 (pdf) ISSN 2489-9860 (print) ISSN 2490-0028 (pdf) http://urn.fi/URN:ISBN:978-952-03-1435-4 PunaMusta Oy – Yliopistopaino Tampere 2020 CONTENTS LIST OF ORIGINAL COMMUNICATIONS ................................................................. 6 ABBREVIATIONS .......................................................................................................... 7 ABSTRACT ..................................................................................................................... 9 TIIVISTELMÄ .............................................................................................................. 11 1 INTRODUCTION ............................................................................................... 13 2 REVIEW OF THE LITERATURE ..................................................................... 15 2.1 Osteoarthritis ............................................................................................. 15 2.1.1 Pathogenesis of Osteoarthritis .................................................. 18 2.1.1.1 Structural changes in OA cartilage ........................... 19 2.1.1.2 Chondrocyte ............................................................. 23 2.1.1.3 Inflammation in OA.................................................. 24 2.1.2 Inflammatory mediators in osteoarthritis .................................. 26 2.1.2.1 Interleukin-1β (IL-1β) .............................................. 27 2.1.2.2 Interleukin-6 (IL-6)................................................... 30 2.1.2.3 Fibroblast growth factor-2 (FGF-2) .......................... 32 2.2 Transient receptor potential ion channels ................................................. 36 2.3 TRPA1 ...................................................................................................... 36 2.3.1 TRPA1 structure and biophysical properties ............................ 37 2.3.2 TRPA1 expression .................................................................... 40 2.3.3 Activators and blockers of TRPA1 ........................................... 41 2.3.4 Functions of TRPA1 ................................................................. 47 2.4 TRPA1 in osteoarthritis ............................................................................ 50 3 AIMS OF THE STUDY ...................................................................................... 53 4 MATERIALS AND METHODS ........................................................................ 54 4.1 Patients (I-IV) ........................................................................................... 54 4.2 Mice (I,III) ................................................................................................ 54 4.3 Cell and cartilage culture .......................................................................... 55 4.3.1 Human osteoarthritic cartilage (IV) .......................................... 55 4.3.2 Human osteoarthritic chondrocytes (I-IV) ............................... 55 4.3.3 Human T/C28a2 chondrocytes (I,II) ........................................ 56 4.3.4 HEK 293 cells (I) ..................................................................... 57 4.3.5 Mouse cartilage culture (I,III) .................................................. 57 4.3.6 Primary murine chondrocytes (III) ........................................... 57 4.4 RNA extraction and quantitative RT-PCR (I-IV) .................................... 58 4.5 Protein extraction and western blotting (I,II) ........................................... 60 4.6 Enzyme-linked immunosorbent assay (ELISA) (I,III,IV) ........................ 61 4.7 Fluo-3-AM measurements (I,II) ............................................................... 61 4.8 Next-Generation RNA-sequencing (NGS) (III) ....................................... 62 4.9 Statistics (I-IV) ......................................................................................... 63 5 SUMMARY OF THE RESULTS ....................................................................... 64 5.1 TRPA1 is functionally expressed in human osteoarthritic chondrocytes (I) ....................................................................................... 64 5.1.1 TRPA1 mRNA is expressed in human chondrocytes .............. 64 5.1.2 TRPA1 mRNA expression is increased by OA-related inflammatory factors ................................................................ 66 5.1.3 Human chondrocytes express a functional TRPA1 channel ..................................................................................... 68 5.2 TRPA1 expression is downregulated by dexamethasone and aurothiomalate in human chondrocytes (II) ............................................. 71 5.3 Effects of TRPA1 in chondrocytes and cartilage (I,III) ........................... 74 5.3.1 TRPA1 mediates IL-6 expression in human OA chondrocytes, and murine cartilage and chondrocytes ............. 74 5.3.2 FGF-2 expression is attenuated by pharmacological inhibition and genetic deletion of TRPA1 in human OA and murine chondrocytes ......................................................... 78 5.3.3 MMP-enzyme and PGE2 production is attenuated by genetic deletion and pharmacological inhibition of TRPA1 ..................................................................................... 80 5.4 Effects of FGF-2 and FGF receptor antagonists in human OA cartilage and chondrocytes (IV) ............................................................... 83 5.4.1 FGF-2 correlates with MMP-enzymes in human OA cartilage explants...................................................................... 83 5.4.2 FGF-2 stimulation exerts catabolic and anti-anabolic responses in human OA chondrocytes ..................................... 85 5.4.3 FGF-2 receptor antagonists induce anabolic and anti- catabolic responses in human OA chondrocytes ...................... 87 6 DISCUSSION ..................................................................................................... 90 6.1 Methodology ............................................................................................ 90 6.2 TRPA1 expression in human chondrocytes ............................................. 93 6.3 Effects of dexamethasone and aurothiomalate on TRPA1 expression .................................................................................................96 6.3.1 Current knowledge on the regulation of TRPA1 expression.................................................................................99 6.4 TRPA1 and production of proinflammatory and catabolic factors.........102 6.4.1 IL-6 .........................................................................................103 6.4.2 FGF-2 .....................................................................................105 6.4.3 MMP-enzymes and PGE2 .......................................................108 6.5 Clinical aspects .......................................................................................109 7 SUMMARY AND CONCLUSIONS................................................................113 8 KIITOKSET (ACKNOWLEDGEMENTS) ......................................................115 9 REFERENCES..................................................................................................117 10 ORIGINAL COMMUNICATIONS.................................................................. 14 3 LIST OF ORIGINAL COMMUNICATIONS This thesis is based on the following original communications: I. Nummenmaa E, Hämäläinen M, Moilanen LJ, Paukkeri EL, Nieminen RM, Moilanen T, Vuolteenaho K, Moilanen E (2016): Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes. Arthritis Res Ther. 18(1):185. II. Nummenmaa E, Hämäläinen M, Moilanen LJ, Moilanen T, Vuolteenaho K, Moilanen E (2017): TRPA1 expression is downregulated by dexamethasone and aurothiomalate in human chondrocytes: TRPA1 as a novel factor and drug target in arthritis. RMD Open. 3(2):e000556. III. Nummenmaa E, Hämäläinen M, Pemmari A, Moilanen LJ, Tuure L, Nieminen RM, Moilanen T, Vuolteenaho K, Moilanen E. TRPA1 as a factor and drug target in osteoarthritis: TRPA1 (transient receptor potential ankyrin 1) mediates interleukin-6 expression in chondrocytes. Submitted for publication. IV. Nummenmaa E, Hämäläinen M, Moilanen