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Interactions Between EGF and the IGF-1 Receptor in Mediating Hgly2-GLP-2-Induced Proliferation in the Murine Small Intestine

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Interactions Between EGF and the IGF-1 Receptor in Mediating Hgly2-GLP-2-Induced Proliferation in the Murine Small Intestine Interactions Between EGF and the IGF-1 Receptor in Mediating hGly2-GLP-2-Induced Proliferation in the Murine Small Intestine by Zivit Fesler A thesis submitted in conformity with the requirements for the degree of Master’s of Science Department of Physiology University of Toronto © Copyright by Zivit Fesler 2019 Interactions Between EGF and the IGF-1 Receptor in Mediating hGly2-GLP-2-Induced Proliferation in the Murine Small Intestine Zivit Fesler Master’s of Science Department of Physiology University of Toronto 2019 Abstract Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal proliferation through downstream mediators. Two known mediators are insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), both essential for the proliferative effect of GLP-2. I hypothesized that pre- and/or co-EGF enhances IGF-1-mediated GLP-2-induced proliferation in the murine model. Chronic combination treatment of EGF and GLP-2 increased intestinal growth in C57Bl/6 mice compared to vehicle, GLP-2 and respective EGF treatments. Treatment of pre-EGF+co-EGF+GLP-2 in intestinal epithelial IGF-1 receptor (IGF-1R) knockout mice restored intestinal growth despite the lack of a receptor essential for GLP-2 signaling. Finally, IGF-1 treatment in organoids did not have an additive effect to EGF on proliferation, but managed to replace EGF as an essential growth factor in the media to maintain normal proliferation. Together, these data suggest that exogenous EGF is an essential mediator for GLP-2 that can supersede the missing signaling pathway of IGF-1R. ii Acknowledgments Graduate school has been an important learning and growing environment for me. It was both joyful and challenging. I met many people who became mentors and good friends and honed my research and critical thinking skills which I will carry on in my future endeavors. There are many people I would like to thank for their support and belief in me as I took this important step. First, I would like to thank my supervisor, Dr. Patricia Brubaker, for her unconditional support and patience as I learned and grew as a researcher. Her continuous mentorship and guidance allowed me to develop invaluable skills that I will carry wherever I go. I would also like to thank my committee member Dr. Dan Drucker for challenging me during committee meetings and providing me with essential tools to better myself as a scientist and a critical thinker. I would like to thank Jennifer Chalmers and Melanie Markovic from the Brubaker lab for their valuable teachings and continuous support of me and my project. Jennifer taught me many of the methods I have mentioned through this thesis, as well as provided me with support through problem-solving, critical thinking, and teaching me how to be an ethical scientist. I want to thank Melanie for providing me with friendship and mentorship throughout my two-year degree, and for teaching me how to be critical of my data, how to analyze it, and how to be a better scientist. I would like to thank Alexandre Hardy from the CFI lab for providing me with much needed training, as well as continuous support during my extensive use of the facility. I would also like to thank Dr. Katie Rowland, who has established the IE-IGF-1R knockout model in our lab, as well as Dr. Robine, Dr. Holzenberger and Dr. Kulkarni for initially providing the transgenic animals. I would like to thank Dr. O’Brien from the University of Toronto and Dr. Kuo from Stanford University for providing me with HEK293 cells for the organoid experiments. iii I would also like to thank the Department of Physiology and the administration, particularly Colleen, Rosalie, and Eva for all their assistance. Finally, I would like to thank my mother, Zila Fesler, and my two close friends, Adrian Esser and Christine Qian for supporting me and continuously pushing me to better myself. I would have not been able to achieve this degree without your emotional support and listening ear. In memory of Bertie Fesler. iv Table of Contents Acknowledgments .................................................................................. iii Table of Contents .................................................................................... v List of Tables ........................................................................................ viii List of Figures ........................................................................................ ix List of Abbreviations .............................................................................. x Introduction .......................................................................... 1 1.1 Rationale ..................................................................................................... 1 1.2 Intestinal Architecture and Proliferation ................................................ 2 1.2.1 Types of cells in the intestine .................................................................................................. 2 1.2.2 Lgr5 and Quiescent Stem Cells ............................................................................................... 3 1.3 Glucagon Like Peptide 2 ........................................................................... 5 1.3.1 Structure, Expression and Secretion ........................................................................................ 5 1.3.2 Clearance ................................................................................................................................. 6 1.3.3 Effects on the Gastrointestinal Tract ....................................................................................... 6 1.4 GLP-2 Receptor ......................................................................................... 8 1.4.1 Discovery and Location........................................................................................................... 8 1.4.2 Signaling ................................................................................................................................ 10 1.5 Intestinal EGF .......................................................................................... 11 1.5.1 Structure, Expression and Secretion ...................................................................................... 11 1.5.2 Effects of EGF on the Gut ..................................................................................................... 11 1.5.3 EGF Receptors and Signaling ............................................................................................... 13 1.5.4 EGF and GLP-2 ..................................................................................................................... 14 1.6 Intestinal IGF-1 ....................................................................................... 15 v 1.6.1 Structure, Expression and Secretion ...................................................................................... 15 1.6.2 Effects of IGF-1 on the Gut ................................................................................................... 16 1.6.3 IGF-1 receptors and signaling ............................................................................................... 17 1.6.4 IGF-1 and GLP-2................................................................................................................... 17 1.7 The Known Interactions Between EGF and IGF-1 .............................. 19 1.8 Hypothesis and Specific Aims................................................................. 20 Materials and Methods ...................................................... 21 2.1 In Vivo Studies ......................................................................................... 21 2.2 In Vitro Studies ........................................................................................ 22 2.3 RNA Extraction and Analysis ................................................................ 24 2.4 Microscopy ............................................................................................... 25 2.5 Proliferation Analysis .............................................................................. 26 2.6 Statistics .................................................................................................... 27 Results ................................................................................. 28 3.1 Determining the Optimal Timing for EGF-GLP-2 Treatment ........... 28 3.1.1 A Combination Treatment with EGF and GLP-2 Increases Small Intestinal Weight........... 29 3.1.2 All Combination Treatments with EGF and GLP-2 Increase Crypt-Villus Height .............. 31 3.1.3 GLP-2 Increases Proliferation while EGF Decreases Apoptotic Markers ............................ 33 3.1.4 Transcripts of Ligands and Their Receptors Did Not Change Between Groups .................. 35 3.2 Investigating the Relationship Between GLP-2, EGF and IGF-1R Using IE-IGF-1R-KO Mice ............................................................................... 37 3.2.1 Validation of the IE-IGF-1R KO in Mice ............................................................................. 38 3.2.2 IE-IGF-1R KO Mice and Control Mice Respond Similarly to Pre-EGF+Co-EGF+GLP-2 Combination Treatment ...................................................................................................................... 39 3.3 Organoids ................................................................................................. 43 3.3.1 qRT-PCR of Organoids Confirms the Absence of GLP-2R and the
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