Western University Scholarship@Western Electronic Thesis and Dissertation Repository 7-20-2015 12:00 AM Investigating The Mechanism Of Ectopic Mineralization In A Mouse Model Of Diffuse Idiopathic Skeletal Hyperostosis (DISH) Neil A. Tenn The University of Western Ontario Supervisor Dr. Cheryle Séguin The University of Western Ontario Joint Supervisor Dr. Jeffrey Dixon The University of Western Ontario Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Neil A. Tenn 2015 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Animal Diseases Commons, Musculoskeletal Diseases Commons, and the Pathological Conditions, Signs and Symptoms Commons Recommended Citation Tenn, Neil A., "Investigating The Mechanism Of Ectopic Mineralization In A Mouse Model Of Diffuse Idiopathic Skeletal Hyperostosis (DISH)" (2015). Electronic Thesis and Dissertation Repository. 2966. https://ir.lib.uwo.ca/etd/2966 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. INVESTIGATING THE MECHANISM OF ECTOPIC MINERALIZATION IN A MOUSE MODEL OF DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH) (Thesis format: Integrated Article) by Neil Alexander Tenn Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Neil Alexander Tenn 2015 Abstract Equilibrative nucleoside transporter 1 (ENT1) transfers adenosine across plasma membranes. Mice lacking ENT1 (ENT1-/-) develop pathological calcification of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Our goal was to investigate the mechanism underlying ectopic mineralization in ENT1-/- mice. We detected aberrant alkaline phosphatase (ALP, promoter of mineralization) activity in the annulus fibrosus (AF) of ENT1-/- mice. In vitro, AF cells from ENT1-/- mice exhibited greater ALP activity than cells from wild-type (WT) mice. Inhibition of ENT1 in the presence of extracellular adenosine modeled in WT cells the phenotype of ENT1-/- cells. We also characterized differences in the AF cell phenotype between WT and ENT1-/- mice using transcriptome analysis, revealing the association between activation of cell cycle and B cells with the induction of mineralization. In summary, both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1-/- mice, possibly through alterations in ALP distribution and activity. Keywords: Adenosine, alkaline phosphatase, equilibrative nucleoside transporters, intervertebral discs, ectopic mineralization II EPIGRAPH “I have not failed. I’ve just found 10, 000 ways that won’t work” Thomas A. Edison III ACKNOWLEDGEMENTS My sincerest thanks go to my supervisor, Dr. Cheryle Séguin, who gave me the opportunity to pursue my Masters degree. For the last two years, you provided me with a “home”, which enabled me to mature both as a student and as a person. I will be forever appreciative of your guidance and wisdom throughout my graduate career. I have always admired your ability to make time for your students while balancing what seems like a thousand different things. Your dedication and passion for science is an inspiration; just observing your work ethic challenges me to become a better scientist and harder worker. You are always available to provide constant and consistent help in my endeavours. I will always be grateful for the time you have invested in me. In addition, I would like to thank my co-supervisor, Dr. Jeffrey Dixon, for your scientific guidance, positive outlook, encouragement and advice over the last two years. I have always taken comfort in knowing I could approach you with my scientific problems and leave with my mind at ease. I would also like to thank my committee members, Drs. Stephen Sims, Peter Chidiac and Lina Dagnino, for your input and positive criticism. Your advice helped me to become a better and more prepared student. Special thanks must go to Courtney Brooks and Diana Quinonez, both of which have helped me so much, whether it was experiments, training or just being around to talk to. I will always be grateful for your help during my Masters degree. Your help was invaluable, and I want you both to know how grateful I am for it. I’d also like to thank all of the members of the Séguin lab (Sumeeta Warraich, Hisataka Ii, Matthew McCann, Jake Bedore, Mark Kim and Matthew Veras). Your encouragement, friendship, training and support have helped me get through the last few years, relatively unscathed. Additionally, I would like to thank the members of the Beier lab (Mike Pest, Paxton Moon, and Margaret Sun) for their friendship and interesting conversations. Without these groups of people my graduate school experience would not be the same. Thank you to both Katie Rabicki and Anusha Ratneswaran. In the past few years, I have become closer to the both of you and would not know what I would have done without your help and support. Katie, you have been an excellent friend and roommate, putting up with my shenanigans and always knowing what to say when I am feeling anxious. To Anusha, your level head and advice always gave me another perspective. In our drives back to Toronto, our discussions were always entertaining and I can honestly say I am glad to have met you. IV There is a core group of friends back home who I can honestly call my family. I am indebted to all of you for your support and ability to keep me laughing and in good spirits. All of you have helped me in different ways, whether you knew it or not, and gave me an escape when my life was becoming overwhelming. I would like to end by thanking my loving and supporting family. To my brother, Brian: thank you for always looking out for me throughout my life. Your phone calls to check-up on me are always and will always be appreciated, even though it may not always seem that way. You have helped me to become more independent, and I know that you will always be there for me. To my parents: your unconditional love and support will never go unappreciated. Thank you for always supporting me through all my life choices; I would not have been able to get here without you. Finally, I would like to thank Catherine Tom for her unfailing support and understanding throughout my graduate career. You always encouraged me to pursue my dreams and were always available for me to talk. You kept me grounded through all my ups and downs. You are my best friend, and I do not know what I would do without you. There are no words that can describe the gratitude I have for each and every one of you. Thank you for being in my life. V TABLE OF CONTENTS ABSTRACT ......................................................................................................................................... II EPIGRAPH ......................................................................................................................................... III ACKNOWLEDGEMENTS ........................................................................................................... IV-V TABLE OF CONTENTS .......................................................................................................... VI-VIII LIST OF TABLES ................................................................................................................................ X LIST OF FIGURES ........................................................................................................................ X-XI LIST OF APPENDICES ................................................................................................................... XII LIST OF ABBREVIATIONS .................................................................................................. XIII-XV CHAPTER 1: INTRODUCTION ........................................................................................................ 1 1.1 Nucleobases, Nucleosides and Nucleotides ..................................................................... 2 1.2 Nucleoside Transporters .................................................................................................. 2 1.2.1 Concentrative Nucleoside Transporters (SLC28A) ................................................................ 4 1.2.2 Equilibrative Nucleoside Transporters (SLC29A) ................................................................... 4 1.2.2.1 ENT1 (SLC29A1) .................................................................................................. 6 1.2.2.2 ENT2 (SLC29A2) .................................................................................................. 6 1.2.2.3 ENT3 and ENT4 .................................................................................................... 6 1.3 Adenosine ........................................................................................................................ 7 1.2.1 Adenosine Bioactivity ........................................................................................................... 7 1.4 Connective Tissue ............................................................................................................ 8 1.4.1 Cartilage ..............................................................................................................................