Molecular Mechanism of Podosome Formation and Proteolytic Function in Human Bronchial Epithelial Cells By Helan Xiao A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Physiology University of Toronto ¤ Copyright by Helan Xiao 2009 Molecular Mechanism of Podosome Formation and Proteolytic Function in Human Bronchial Epithelial Cells Helan Xiao Doctor of Philosophy Graduate Department of Physiology University of Toronto 2009 Abstract In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodelling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading ii fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCĮ, from the cytosol to the podosomes, whereas atypical PKCȗ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs. iii Acknowledgements The thesis is to my beloved parents Mr. Qingcai Xiao and Ms. Dong Li. I sincerely appreciate their love and support throughout my life. First and foremost, I would like to give my greatest gratitude to my supervisor Dr. Mingyao Liu, who has provided me opportunity, knowledge, mentorship and assistance in all these six years. As an international student and non-native English speaker, I met numerous obstacles when I first came to Toronto. Dr. Liu always encourages me and tries his best to cheer me up whenever I had troubles. Thanks to all of my current and previous academic committee members, Dr. Andras Kapus, Dr. Alan S. Mak, Dr. Wei-Yang Lu, Dr. John F. MacDonald and Dr. Daniela Rotin, for their continuous guidance, help, support, thoughtful discussion, friendship and inspiration. I will always remember their serious scientific attitudes, passion for research, dedication to science, and industrious work. I would like to give my special gratitution to Dr. Shaf Keshavjee, Dr. Thomas Waddell, Dr. Marc de Perrot and Dr. David Hwang for their support, leadership and help. I would like to especially express my thanks to Dr. Jing Xu, Dr. Monika Lodyga, Dr. Feng Xu, Dr. Bing Han, Ms. Xiao-Hui Bai, and Dr. Jeya Nadesalingam who have provided me their technical expertise, suggestion, kindly assistance, thoughtful discussion, encouragement and friendship. Special thanks to Ms. Xiao-Hui Bai to teach me Real-time RT-PCR and PKC assay, to Mr. Rob Eves to teach me in situ zymography and gelatin gel zymography assay. I am also fortunate the dear friendship, help and support from all of my current and previous lab members: Mr. Peter S. Tang, Dr. Atsushi Shiozaki, Ms. Yu Zhang, Dr. Xiaolin He, Dr. Marco Mura, Dr. Takeshi Oyaizu, Ms. Roli Bawa, Mr. Matthew Rubacha, Dr. Yongfang Yuan, Dr. Shane Fung and Ms. iv Ivone Ornelas. Many thanks to the help from all of my summer students: Mr. David Laugren, Mr. Wayne Kan, Ms. Christina Yep, Ms. Debbie Li, and Mr. Wasim Kagzi. I would give my special acknowledgements to our colaboraters in Queen’s University for their contributions to my publications, technical support, contributive and thoughtful discussions: Dr. Alan S. Mak, Mr. Rob Eves, Ms. Lily Jia, and Dr. Bradley Webb. Great thanks to our entire research team members and local collaborators: Dr. Lowell Langille, Ms. Hanna Zhihong Yun, Dr. Amy Wong, Dr. Masaaki Sato, Dr. Kota Ishizawa, Dr. Masashi Gotoh, Dr. Shin Hirayama, Dr. Licun Wu, Dr. Macelo Cypel, Dr. Masaki Anruku, Mr. Pascal Dunchesneau, Mr. Paul Chartrand, Dr. Rongyu Jin, Dr. Yingzhe Zhou, Dr. Peter Sabatini, Mr. Marc L. Chretien, Dr. Rosalind Silverman and Dr. Lorelei Silverman. I would like to give my sincere gratitude to the current and previous graduate coordinators, graduate administrators and graduate assistants in Department of Physiology for their help, kind suggestion, consultant and encouragement, Dr. Milton P. Charlton, Dr. Martin Wojtowicz, Dr. Denise Belsham, Ms. Julie Weedmark, Ms. Rosalie Pang and Ms. Colleen Shea. Great thanks to all professors and teaching assistants in the courses I have taken. And many thanks to all the people who ever kindly helped me during my PhD study. I am also grateful for the financial support from Peterborough K. M. Hunter Graduate Studentship for cancer research. v Table of Contents ABSTRACT.................................................................................................................................. II ACKNOWLEDGEMENTS .......................................................................................................IV TABLE OF CONTENTS ...........................................................................................................VI LIST OF FIGURES .................................................................................................................. XII LIST OF TABLES ...................................................................................................................XVI LIST OF ABBREVIATIONS ............................................................................................... XVII LIST OF PUBLICATIONS .................................................................................................XXIII 1. CHAPTER ONE. GENERAL BACKGROUND - EPITHELIAL CELL MIGRATION IN HUMAN LUNG PHYSIOLOGY, PATHOPHYSIOLOGY AND RESPIRATORY DISEASES ..................................................................................................................................... 1 1.1 Overview - Lung epithelial cell migration ........................................................................ 3 1.2 Respiratory epithelial cell migration is an early event in the repair process................ 4 1.2.1 Epithelial cells in the airway and lung parenchyma................................................. 4 1.2.2 Airway epithelial cell migration occurs after tissue injury as an early event ........ 6 1.3 Regulation of airway epithelial cell migration ............................................................... 10 1.4 Alveolar epithelial cell migration occurs after tissue injury......................................... 13 vi 1.5 Perspective: insight from epithelial cell migration to understanding of injury and repair of lung diseases ............................................................................................................ 15 2. CHAPTER TWO. GENERAL BACKGROUND - CELLULAR STRUCTURES FOR CELL ADHESION, MIGRATION AND INVASION ............................................................ 17 2.1 Molecular mechanisms of cell migration ........................................................................ 18 2.2 Focal adhesion ................................................................................................................... 21 2.3 Podosome ........................................................................................................................... 24 2.4 Invadopodia....................................................................................................................... 28 2.5 Molecular components of podosomes.............................................................................. 28 2.5.1 Src................................................................................................................................ 29 2.5.2 PI3K/Akt..................................................................................................................... 31 2.5.3 MAPKs........................................................................................................................ 34 2.5.4 Matrix metalloproteinases........................................................................................