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Functional Interactions Between the Discoidin Domain Receptor 1 and Β1 Integrins

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Functional Interactions Between the Discoidin Domain Receptor 1 and Β1 Integrins FUNCTIONAL INTERACTIONS BETWEEN THE DISCOIDIN DOMAIN RECEPTOR 1 AND β1 INTEGRINS by Lisa Alexandra Staudinger A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Dentistry University of Toronto © Copyright by Lisa Alexandra Staudinger 2013 Functional Interactions Between the Discoidin Domain Receptor 1 and β1 Integrins Lisa Alexandra Staudinger Master of Science Graduate Department of Dentistry University of Toronto 2013 Abstract The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated. ii Acknowledgments My Master of Science has been the most challenging, most tiring, but by far, the most rewarding two years of my life. However, I cannot take sole credit for the completion of my degree, as this work would not have been possible if not for the support of so many wonderful individuals. First and foremost, I would like to thank Dr. McCulloch for taking a chance and allowing me to work in his lab. His immense intelligence and passion for science have been an inspiration, and his guidance and support, invaluable. I have felt it a privilege from day one to be working under his supervision. I am more than grateful to Dr. Moriarty for being endlessly generous with her time and for always providing me with valuable advice and direction. I am thankful to my committee member Dr. Bendeck for her more than helpful suggestions. Thank you to Wilson Lee for always making me feel like the funniest person alive by laughing at my jokes, no matter how bad they were, and of course, for the endless hours he has put in to help make my thesis complete. For much of the contents of this work I must also thank Stephen Spano, working with him was always a blast. I would like to say thanks to Carol Laschinger for teaching me how to make the perfect western blot and to my lab-mates Hamid Mohammadi, Dhaarmini Rajshankar, Qin Wang, Vanessa Pinto, Dominik Fritz, Ilana Talior and Pam Arora for their advice, friendship and fun times in the lab. I would like to thank Andrew Peters for inspiring me to not be afraid to pursue science, for keeping me motivated, and for his constant support and encouragement. Last but not least, I would like to thank my family. My grandparents Grete and Peter for their financial, and of course emotional investment in my future, and to my brother PJ and parents Gail and Herb for their unconditional love and support. iii Table of Contents Abstract ………………………………………………………………………….... ii Acknowledgements ……………………………………………………….……… iii Table of Contents …………………………………………………………………. iv List of Diagrams …………………………………………………………………… vii List of Figures …………………………………………………………………….. viii List of Appendices ………………………………………………………………... xii Abbreviations …………………………………………………………………….. xiii Chapter 1 – Literature Review ………………………………………………….. 1 I. Soft Connective Tissue ……………………………………………….. 1 A. Structure and Function of the Extracellular Matrix ...…………….... 1 i. Collagens …………………………………………………… 2 B. Cells of the Soft Connective Tissue ………………………………… 3 II. Homeostasis …………………………………………………………… 3 A. Function ……………...…………………………………………….. 4 B. Regulatory Mechanisms…………………………………………….. 4 i. Matrix Metalloproteinases…………………………………... 5 III. Disorders of Homeostasis …………………………………………….. 6 A. Mechanisms of Fibrosis…………………………………………….. 6 B. Drug-Induced Gingival Overgrowth………………………………… 7 iv i. Cyclosporin A-Induced Gingival Overgrowth……………… 8 IV. Phagocytosis …………………………………………………………… 9 A. Mechanism of action………………………………………………… 9 V. Receptors ……………………………………………………………… 12 A. Collagen binding: The Rate Limiting Step in Matrix Remodeling…. 12 B. β1 Integrins…………………………………………………………. 13 i. Structure and Function……………………………………… 13 ii. Integrin Signaling and Regulation of Activity……………… 15 iii. Glycosylation……………………………………………….. 16 C. DDR1……………………………………………………………….. 18 i. Structure and Function……………………………………… 18 ii. Isoforms…………………………………………………….. 20 iii. Signaling Pathways…………………………………………. 21 iv. DDR1 and Fibrosis…………………………………………. 22 VI. DDR1 Interactions ……………………………………………………. 23 A. DDR1 and Metalloproteinases……………………………………… 23 B. DDR1 and β1 Integrins……………………………………………… 24 Statement of the Problem ……………………………………………………….... 26 Chapter 2 – Introduction ………………………………………………………… 27 Materials and Methods……………………………………………………………. 30 Results ……………………………………………………………………………… 37 v Discussion ………………………………………………………………………….. 45 Figure Legends ……………………………………………………………………. 51 Figures ……………………………………………………………………………... 57 Future Directions and Conclusions…………………………………………….… 68 Appendix …………………………………………………………………………... 71 Appendix Legends ………………………………………………………………… 72 Appendix Figures…………………………………………………………………... 74 References …………………………………………………………………….…… 77 vi List of Diagrams Diagram 1. A severe case of Cyclosporin A-induced gingival overgrowth…….. 9 Diagram 2. Intracellular pathway of collagen degradation: Phagocytosis…….... 10 Diagram 3. The β1 integrin family of extracellular matrix receptors …………… 14 Diagram 4. β1 integrin activation in response to collagen binding……………… 16 Diagram 5. Structure of DDR1 transcript variants and isoforms …………..…… 21 vii List of Figures Figure 1. ……………………………………………………………………… 57 A. Immunoblot of DDR1 expression B. Immunoblot of β1 integrin expression Figure 2. ……………………………………………………………………… 58 A. Histogram of collagen-coated bead binding after 1 hour B. Histogram of collagen-coated bead binding after 2, 4 and 24 hours C. Histogram of fibronectin-coated bead binding after 1 hour D. Histogram of collagen-coated bead binding after incubation with CD29 blocking antibody E. Histogram of cell surface α2 integrin expression Figure 3. ……………………………………………………………………… 59 A. Histogram of floating gel collagen contraction assay B. Histogram of attached gel contraction assay Figure 4. ……………………………………………………………………… 60 A. Images of in vitro cell migration assay B. Line plot of reduction in scratch width viii C. Histogram of mean slope of scratch width Figure 5. ……………………………………………………………………… 61 A. TIRF images of activated β1 integrin staining in focal adhesions B. Histogram of stained β1 integrin total cell area C. Histogram of number of activated β1 integrin containing focal adhesions D. Histogram of stained activated β1 integrin focal adhesion length E. Histogram of stained area of activated β1 integrin containing focal adhesions Figure 6. ……………………………………………………………………… 62 A. TIRF images of talin stained focal adhesions B. Histogram of stained talin total cell area C. Histogram of number of stained talin containing focal adhesions D. Histogram of stained talin focal adhesion length E. Histogram of stained area of talin containing focal adhesions Figure 7. ……………………………………………………………………… 63 A. TIRF images of paxillin stained focal adhesions B. Histogram of paxillin stained total cell area ix C. Histogram of number of paxillin containing focal adhesions D. Histogram of stained paxillin focal adhesion length E. Histogram of stained area of paxillin containing focal adhesions Figure 8. ……………………………………………………………………… 64 A. TIRF images of vinculin stained focal adhesions B. Histogram of vinculin stained total cell area C. Histogram of number of vinculin containing focal adhesions D. Histogram of stained vinculin focal adhesion length E. Histogram of stained area of vinculin containing focal adhesions Figure 9. ……………………………………………………………………… 64 A. Histogram of total β1 integrin surface expression B. Histogram of activated β1 integrin expression of cells plated on collagen C. Histogram of activated β1 integrin expression of cells plated plastic D. Histogram of activated β1 integrin expression of cells in suspension Figure 10. Immunoblot of β1 integrin expression after treatment with the disintegrin jararhagin…………………………………………………………… 66 x Figure 11. Immunoblot of β1 integrin expression after treatment with endoglycosidase H …………………………………………………………………….... 67 xi List of Appendices Appendix 1 …………………………………………………………….... 74 A. Histogram of collagen-coated bead binding in cyclosporin A treated human gingival fibroblasts after 1 hour B. Histogram of collagen-coated bead binding in cyclosporin A treated cells after 1 hour Appendix 2 …………………………………………………………….... 75 A. Histogram of collagen-coated bead binding in cyclosporin A treated cells after overnight plating inside collagen gels B. Histogram of floating collagen gel contraction assay of cyclosporin A treated cells C. Histogram of attached collagen gel contraction assay of cyclosporin A treated cells Appendix 3. ………………………………………………………………. 76 A. Line plot of reduction in scratch width of cyclosporin A treated cells B. Histogram of mean slope of scratch width of cyclosporin
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