LAURI MOILANEN TRPA1 in Inflammation TRPA1 LAURI MOILANEN Acta Universitatis Tamperensis 2173 LAURI MOILANEN TRPA1 in Inflammation AUT 2173 AUT LAURI MOILANEN TRPA1 in Inflammation ACADEMIC DISSERTATION To be presented, with the permission of the Board of the School of Medicine of the University of Tampere, for public discussion in the small auditorium of building B, School of Medicine, Medisiinarinkatu 3, Tampere, on 10 June 2016, at 12 o’clock. UNIVERSITY OF TAMPERE LAURI MOILANEN TRPA1 in Inflammation Acta Universitatis Tamperensis 2173 Tampere University Press Tampere 2016 ACADEMIC DISSERTATION University of Tampere, School of Medicine Tampere University Hospital National Doctoral Programme of Musculoskeletal Disorders and Biomaterials (TBDP) Finland Supervised by Reviewed by Docent Riina Nieminen Docent Paula Rytilä University of Tampere University of Helsinki Finland Finland Professor Lauri Lehtimäki Professor Ulf Simonsen University of Tampere Aarhus University Finland Denmark Professor Eeva Moilanen University of Tampere Finland The originality of this thesis has been checked using the Turnitin OriginalityCheck service in accordance with the quality management system of the University of Tampere. Copyright ©2016 Tampere University Press and the author Cover design by Mikko Reinikka Distributor: [email protected] https://verkkokauppa.juvenes.fi Acta Universitatis Tamperensis 2173 Acta Electronica Universitatis Tamperensis 1672 ISBN 978-952-03-0131-6 (print) ISBN 978-952-03-0132-3 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://tampub.uta.fi Suomen Yliopistopaino Oy – Juvenes Print Tampere 2016 441 729 Painotuote To my close ones. Contents List of Original Communications ..................................................................................... 9 Abbreviations ..................................................................................................................... 10 Abstract ............................................................................................................................... 11 Tiivistelmä ........................................................................................................................... 13 Introduction ....................................................................................................................... 15 1 Review of the Literature .......................................................................................... 19 1.1 Transient Receptor Potential Ion Channels ................................................. 19 1.2 TRPA1 ............................................................................................................... 23 1.2.1 Trpa1 Gene and Protein Structure ....................................................... 23 1.2.2 TRPA1 Expression ................................................................................. 25 1.2.3 TRPA1 Electrophysiology ..................................................................... 26 1.2.4 Exogenous Chemical Activators of TRPA1 ....................................... 27 1.2.5 Endogenous Chemical Activators of TRPA1 ..................................... 29 1.2.6 Activation of TRPA1 by Cold and Mechanical Stimuli ..................... 30 1.2.7 Blockers of TRPA1 ................................................................................. 31 1.2.8 Physiological Function of TRPA1 ........................................................ 32 1.2.9 Non-Neuronal TRPA1 ........................................................................... 34 1.2.10 TRPA1 in Pain ......................................................................................... 34 1.2.11 TRPA1 in Inflammation ........................................................................ 39 1.3 Inflammation .................................................................................................... 40 1.4 Carrageenan-Induced Acute Paw Inflammation ......................................... 42 1.5 Gout ................................................................................................................... 43 1.6 Monosodium Urate-Induced Experimental Gout ...................................... 44 1.7 Osteoarthritis .................................................................................................... 45 1.8 Monosodium Iodoacetate-Induced Experimental Osteoarthritis and Inflammation ..................................................................................................... 46 1.9 Allergic Inflammation ...................................................................................... 48 1.10 Ovalbumin-Induced Experimental Allergic Inflammation ........................ 50 1.11 Pinosylvin ........................................................................................................... 51 2 Aims of the Study ...................................................................................................... 52 3 Materials and Methods ............................................................................................. 53 3.1 Compounds ....................................................................................................... 53 3.2 Mice .................................................................................................................... 53 3.3 Acute Paw Edema ............................................................................................ 55 3.4 Monosodium Urate-Induced Acute Arthritis............................................... 55 3.5 Monosodium Urate-Induced Air-Pouch Inflammation ............................. 56 3.6 Monosodium Iodoacetate-Induced Osteoarthritis ...................................... 56 3.7 Ovalbumin-Induced Conjunctivitis ............................................................... 57 3.8 Cell and Cartilage Culture ............................................................................... 58 3.8.1 HEK 293 Cells ......................................................................................... 58 3.8.2 Human Osteoarthritic Chondrocytes ................................................... 59 3.8.3 Mouse Cartilage Culture ......................................................................... 59 3.8.4 Eosinophil Isolation, Culture and Viability Determination .............. 59 3.8.5 Splenocyte Isolation and Interleukin-13 Measurements .................... 61 3.9 Protein Extraction and Western Blotting ..................................................... 61 3.10 Enzyme Linked Immunosorbent Assay ....................................................... 62 3.11 RNA Extraction and Reverse Transcription Polymerase Chain Reaction .. ............................................................................................................................. 62 3.12 Patch Clamp, Fura-2-AM and Fluo-3-AM Measurements ........................ 64 3.12.1 Patch Clamp Measurements ................................................................... 64 3.12.2 Fura-2-AM Measurements ..................................................................... 64 3.12.3 Fluo-3-AM Measurements ..................................................................... 65 3.13 Statistics .............................................................................................................. 65 4 Summary of Results .................................................................................................. 66 4.1 Role of TRPA1 in Carrageenan-Induced Acute Inflammation (Study I) 66 4.1.1 TRPA1 Activation Enhances Carrageenan-Induced Paw Edema ... 66 4.1.2 TRPA1 Mediates AITC-Induced Edema in a COX-Dependent Manner ...................................................................................................... 67 4.1.3 AITC Increases COX-2 Expression in a TRPA1-Dependent Manner ...................................................................................................... 68 4.1.4 Carrageenan is not a Direct TRPA1 Activator ................................... 69 4.2 TRPA1 in Experimental MSU Crystal-induced Gouty Inflammation (Study II) ............................................................................................................ 69 4.2.1 TRPA1 Enhances MSU Crystal-Induced Gouty Inflammation ...... 69 4.2.2 TRPA1 Mediates Pain in Experimental Gouty Arthritis ................... 72 4.2.3 MSU Crystals do not Activate TRPA1 Directly ................................. 73 4.3 Involvement of TRPA1 in MIA-Induced Experimental Osteoarthritis and Inflammation (Study III) ......................................................................... 73 4.3.1 MIA-Induced Acute Inflammation is Partly Dependent on TRPA1 Activation ................................................................................................. 73 4.3.2 MIA Increases COX-2 Expression in Chondrocytes via TRPA1 ... 76 4.3.3 TRPA1 Mediates Cartilage Degeneration and Joint Pain in MIA- Induced Osteoarthritis ............................................................................ 79 4.3.4 MIA is not a Direct TRPA1 Activator ................................................. 81 4.4 TRPA1 in Ovalbumin-Induced Acute Allergic Inflammation (Study IV) .. ............................................................................................................................. 81 4.4.1 Activation of TRPA1 is Crucial in the Development of Ovalbumin- Induced Allergic Conjunctivitis ............................................................. 81 4.4.2 TRPA1 does not Affect Eosinophil