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Regulation of Claudin-3 Expression in Kidney Tubular Epithelial Cells

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Regulation of Claudin-3 Expression in Kidney Tubular Epithelial Cells Regulation of Claudin-3 Expression in Kidney Tubular Epithelial Cells by Shaista Anwer A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Shaista Anwer 2020 Regulation of Claudin-3 Expression in Kidney Tubular Epithelial Cells Shaista Anwer Master of Science Institute of Medical Science University of Toronto 2020 Abstract The overall objective of my studies was to gain insight into the regulation of the tight junction protein, claudin-3, upon inflammatory stimuli in kidney tubular epithelial cells. Claudins mediate paracellular transport and modulate key cellular events like proliferation, migration and differentiation. The inflammatory cytokine Tumor Necrosis Factor-α (TNFα) is a pathogenic factor in kidney disease and alters epithelial permeability. However, the effect of TNFα on claudin-3 expression in the tubules and the mechanisms are not defined. My studies showed that TNFα elevated claudin-3 expression in kidney tubular cells. This effect was due to increased claudin-3 synthesis and mediated by two signaling pathways: extracellular signal regulated kinase- dependent activation of NFκB and protein kinase A-induced CREB1 activation. Claudin-3 overexpression elevated transepithelial resistance in tubular cells and it may play a role in regulating tubular epithelial permeability. Claudin-3 downregulation also affected cell cycle proteins; thus, claudin-3 may also affect cell proliferation. ii Acknowledgements I would like to dedicate this thesis to two important people in my life: my mother, Tahniyat Anwer and my supervisor, Dr. Katalin Szászi. My mom has been a great source of inspiration and a pillar of strength in my life and has always supported me in my endeavours. I will be forever grateful for all her hard work and the sacrifices she has made throughout her life. I would also like to acknowledge my supervisor, Dr. Katalin Szászi, for her constant encouragement and guidance throughout my Master’s. She has played a major role in my academic and personal growth and she has definitely inspired me to be a well-rounded researcher. I am extremely grateful that I had this opportunity to work with her. It would have been impossible to write this thesis without her encouragement and assistance. I would also like to thank my dad and my sisters for always supporting me and for understanding my dedication and passion for research. I would also like to thank my grandparents, uncles, aunts and cousins for always wishing me the best and congratulating me for all my achievements. I would also like to thank my close friends for cheering me up and constantly supporting me. I would also like to express my gratitude to my program advisory committee members, Dr. András Kapus and Dr. Mauricio Terebiznik, for their valuable advice and guidance throughout my Master’s. I really appreciate their input and their passion for science which has fueled my curiosity for many aspects of my project. I would also like to thank all the current and previous lab members, including Qinghong Dan, Shruthi Venugopal, Emily Branchard, Iris Huang, Jenny Xiao and Vida Maksimoska, for helping me with my project. I have had a very enjoyable experience of working with all of them. I have especially learnt a lot from Qinghong Dan and Shruthi Venugopal and I am thankful that I had the opportunity to work with such excellent researchers. I would also like to thank members of the Kapus lab, Pam Speight, Michael Kofler and Zena Miranda, for their kindness, positivity and for providing a different perspective of life. I am also thankful for Muskan Gupta, Zahra Khan, Misha Ditmans and Samantha Mahabir for creating a positive, lively and friendly atmosphere in our office. They were definitely a great addition to our office, and I had a great time having endless conversations with them. Finally, I am also really appreciative of the continuous support of all the people on the 6th floor as well as the core facility of the Li Ka Shing Knowledge Institute. iii Statement of Contributions I am the primary author of this thesis. I have participated in the planning, execution and analysis of research experimentation. I have received funding from UofT Open Fellowship and St. Michael’s Hospital Research Training Centre Scholarship. I would also like to acknowledge the contributions of my supervisor Dr. Katalin Szászi and my program advisory committee (PAC) members, Dr. András Kapus and Dr. Mauricio Terebiznik. My supervisor and PAC members provided tremendous mentorship and guidance in planning, executing, analysing and interpreting my experiments and experimental findings. I would also like to acknowledge the contributions of the following individuals: Qinghong Dan, senior lab technician – provided assistance with some of my experiments. Emily Branchard, past lab member – made initial observations for this project. Caterina Di Ciano-Oliveira, bioimaging specialist – provided microscopy training and assisting with acquisition of confocal images and 3D analysis of confocal images. Vida Maksimoska, volunteer – provided assistance with some of my experiments. iv Table of Contents Abstract.......................................................................................................................ii Acknowledgements.....................................................................................................iii Statement of Contributions.........................................................................................iv List of Abbreviations...................................................................................................vii List of Figures..............................................................................................................ix List of Tables...............................................................................................................ix List of Publications.......................................................................................................x CHAPTER 1 Literature Review.......................................................................................1 1.1 Epithelial cells....................................................................................................1 1.2 Intercellular junctional complexes.......................................................................1 1.3 Tight junctions: structure and function.................................................................4 1.3.1 Tight junction structure.................................................................................4 1.3.2 Overview of tight junction functions: gate, fence and signaling…......................4 1.4 Tight junction proteins........................................................................................5 1.4.1 Claudin proteins......................................................................................5 a. Claudin structure…....................................................................................5 b. Functions of claudins.................................................................................8 c. Claudin localization and expression............................................................9 d. Claudin and tight junction regulation..........................................................9 e. Role of claudins in diseases......................................................................11 1.4.1.1 Claudin-3 a. Claudin-3 structure, expression and interactions................................12 b. Functions of claudin-3.......................................................................14 c. Claudin-3 expression regulation.........................................................18 d. Role of claudin-3 in diseases..............................................................24 v 1.5 Inflammatory cytokines and TJs.......................................................................27 1.5.1 TNFα structure and synthesis.......................................................................27 1.5.2 TNFα receptor activation and signaling pathways..........................................28 1.5.3 Role of TNFα in kidney diseases....................................................................32 CHAPTER 2 Research Aims/Objectives and Hypotheses..............................................33 CHAPTER 3 Materials and Methods............................................................................36 CHAPTER 4 TNFα-induced claudin-3 expression regulation in kidney tubular epithelial cells............................................................................................................41 Results................................................................................................................41 4.1 TNFα induced an increase in Cldn-3 protein expression.......................................41 4.2 TNFα did not alter Cldn-3 degradation and increased Cldn-3 mRNA levels............44 4.3 TNFα-induced increase in Cldn-3 requires NFκB and CREB1 and not Slug..............46 4.4 TNFα-induced increase in Cldn-3 requires ERK-mediated NFκB activation.............51 4.5 TNFα-induced elevation in Cldn-3 requires PKA-dependent CREB1 activation.......54 4.6 Sp1 regulates TNFα-induced Cldn-3 upregulation................................................57 4.7 Cldn-3 overexpression increased transepithelial resistance.................................59 4.8 Cldn-3 silencing upregulates the cell cycle protein p27kip1.................................62
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