PROVISIONAL EXTRACELLULAR MATRIX CITRULLINATION AS A STIMULUS FOR PATHOLOGICAL FIBROBLAST ACTIVATION A Dissertation Presented to The Academic Faculty By Victoria Lauren Stefanelli In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University May of 2018 COPYRIGHT © 2018 BY VICTORIA LAUREN STEFANELLI PROVISIONAL EXTRACELLULAR MATRIX CITRULLINATION AS A STIMULUS FOR PATHOLOGICAL FIBROBLAST ACTIVATION Approved by: Dr. Thomas Barker, Advisor Dr. Melissa Kemp School of Biomedical Engineering School of Biomedical Engineering University of Virginia Georgia Institute of Technology Dr. Johnna Temenoff Dr. Eric White School of Biomedical Engineering Medical School Georgia Institute of Technology University of Michigan Dr. Matthew Torres School of Biological Sciences Georgia Institute of Technology Date Approved: March 29th, 2018 ACKNOWLEDGEMENTS The completion of this work was truly a group effort. I would like to start by thanking all those who provided technical and scientific assistance, especially including the following post-doctoral and graduate student mentors: Ashley Brown, Alison Douglas, Dwight Chambers, John Nicosia, Vincent Yeh, Ping Hu, and Leandro Moretti. For literally working alongside to assist in various animal and benchtop experiments, even when it was outside the scope of their own responsibilities, I would like to acknowledge Vincent Yeh, Dwight Chambers, Mary Harp, Dan Abebayehu, and Riley Hannan. I’m also very grateful to my two awesome undergraduate students Kelly Pesson and Tommy Ng for being so diligent in their work and making my life much easier in the process. And, of course, this work would not have been shaped into its current form without the invaluable scientific feedback of my committee members including Dr. Tom Barker, Dr. Johnna Temenoff, Dr. Eric White, Dr. Matthew Torres, and Dr. Melissa Kemp. Certainly, “any road followed precisely to its end leads precisely nowhere” (Dean Herbert). The goal of my graduate studies was to prepare myself for the next stage of my scientific career, and so I am also grateful to everyone who assisted in my career exploration and development these past few years. To this end I would like to give a special thanks to my mentor Tom Barker who contributed greatly to my growth as an independent researcher by allowing me to pursue a project outside the scope of our lab’s existing research and to truly make it my own. Further, he granted me permission to take three months off for an industrial internship as well as to attend conferences strictly for iii the goal of professional development. I also need to thank my boss at Amgen, Dr. Abraham Gurerro for his instrumental professional guidance, as well as everyone at Amgen who took the time to offer both life and scientific advice. Finally, I want to thank Sally Gerrish, the Emory BEST program, and the Georgia Tech Career Development Center for sponsoring excellent guest speakers and workshops that provided insights into every aspect of scientific careers and career preparation that I could have possibly wanted. Finally, I need to acknowledge that the journey that has become my thesis work has not been without its struggles. In fact, I have never failed so many times at so many things as during my time as a graduate student. Of course, if “success is going from failure to failure without a loss of enthusiasm” (Winston Churchill) I have many to thank for helping to maintain my sanity and at least a modicum of enthusiasm for my work throughout graduate school. Special mentions are necessary to Dwight Chambers, Jessica Joyce, Erin Hannen Edwards, Betsy Campbell, Cheryl Lau Emeterio, Travis Meyer, Claire Segar Olingy, Thien Tran, Amy Su, and Daniel Brown. And last but not least, I need to thank my family, in particular my parents and my brother who offered me endless love and support throughout this journey. Despite their own busy lives, they somehow always found the time for phone calls and offered nothing but empathy, even in face of my tendency to ramble on about research woes for which I’m sure they had little real interest. After all, “no young person on earth is so excellent in all respects as to need no uncritical love” (Kurt Vonnegut). iv TABLE OF CONTENTS ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF SYMBOLS AND ABBREVIATIONS xi SUMMARY xiii CHAPTER 1. Introduction and Specific Aims 1 CHAPTER 2. Background Information 4 2.1 Citrullination in Human Health and Disease 4 2.1.1 What is Citrullination? 4 2.1.2 Anti-Citrullinated Peptide Antibodies 4 2.1.3 HOW does Citrullination Occur: An Introduction to Peptidyl Arginine Deiminase Enzymes and their Function 6 2.1.4 WHERE Does Citrullination Occur 8 2.1.5 The Role of Citrullination in Disease 14 2.1.6 Citrullination Inhibition Efforts 24 2.1.7 The Physiological Function of PAD Enzymes and Citrullination 28 2.2 Activated Fibroblasts 30 2.2.1 Rheumatoid Arthritis Background 31 2.2.2 Activated Fibroblasts in Rheumatoid Arthritis 32 2.2.3 Activated Fibroblasts in Fibrosis 37 2.2.4 Activated Fibroblasts in Cancer 40 2.3 Integrin-Mediated Mechanotransduction 43 2.3.1 Mechanotransduction Signalling 44 CHAPTER 3. The Influence of Citrullinated Fibronectin on Integrin Binding and Downstream Signaling 50 3.1 Abstract 50 3.2 Introduction 51 3.3 Materials and Methods 54 3.3.1 Protein Citrullination 54 3.3.2 Coating Coverslips with Protein 55 3.3.3 Cell Culture 55 3.3.4 COLDER Assay for Citrullination Verification 56 3.3.5 Dot Blot Citrullination Verification 56 3.3.6 In-Gel Protein Digestion for Mass Spectrometry 56 3.3.7 Mass Spectrometry 57 3.3.8 Interferometry 58 3.3.9 CHO Cell Adhesion Assays 58 v 3.3.10 Focal Adhesion Complex Staining 59 3.3.11 Rac and Rho GLISAs 60 3.3.12 Magnetic Bead Force-Inducible Co-Immunoprecipitation 61 3.3.13 Statistical Analysis 62 3.4 Results 62 3.4.1 Protein Citrullination can be Confirmed as a Dose-Dependent Function of PAD Concentration 62 3.4.2 Mass Spectrometry Identifies 24 Unique Sites of Fn Citrullination 64 3.4.3 Citrullination of Fn Decreases αvβ3 Adhesion and Has Minor Impacts on α5β1 Attachment 66 3.4.4 Citrullination of Fn Results in a αvβ3 to α5β1 Integrin Switch 71 3.4.5 Citrullination of Fn Causes Force-Sensitive Upregulation of FAK-SRC-ILK- GSK Signalling 74 3.4.6 Citrullination of Fn Results in Increases of Mechano-responsive proteins F- actin and Vinculin, but not Rac or Rho 76 3.5 Discussion 78 3.5.1 Interpretation of Mass Spectrometry Results 78 3.5.2 Evidence for a αvβ3 to α5β1 Integrin Switch and its Potential Implications 84 3.6 Conclusion 90 CHAPTER 4. The influence of Citrullinated Provisional Extracellular Matrix on Fibroblast phenotype 92 4.1 Abstract 92 4.2 Introduction 92 4.3 Materials and Methods 95 4.3.1 Adhesion and Cell Morphology Assays 95 4.3.2 9*10 Cell Adhesion 95 4.3.3 AFM analysis of Cell Stiffness 96 4.3.4 Apoptosis 96 4.3.5 Cell Proliferation Assays 97 4.3.6 Cell Metabolism Assays 98 4.3.7 Strained Gel Contraction Assay 98 4.3.8 Floating Gel Contraction Assay 99 4.3.9 α-actinin Analysis 100 4.3.10 Real-time Paxillin Turnover Analysis 100 4.3.11 Random Migration Assays 101 4.3.12 Wound Healing Assays 101 4.3.13 Statistical Analysis 102 4.4 Results 102 4.4.1 Fibroblast Adhesion and Spreading is Reduced on Cit Fn 102 4.4.2 Fibroblast Proliferation, Metabolism, and Apoptotic Resistance are Not Impacted by Cit Fn 104 4.4.3 Fibroblast Stiffness is Enhanced on Cit Fn 106 4.4.4 Fibroblasts Possess a Diminished Capacity to Contract Citrullinated Bulk Matrix 107 4.4.5 Fibroblasts Display Increased Focal Adhesion Turnover on Cit Fn 109 4.4.6 Fibroblast Random and Directional Migration is Enhanced on Cit Fn 112 vi 4.5 Discussion 114 4.5.1 Interpretation of Enhanced Migration on Cit Fn 118 4.6 Conclusions 121 CHAPTER 5. Overall Conclusions and Future Directions 123 APPENDIX A. 133 A.1 Definitions and Abbreviations 133 5.1 Additional Interferometry Results 134 REFERENCES 136 vii LIST OF TABLES Table 1 Signaling Proteins 48 Table 2 Citrullination Sites and their locations within Fn 80 Regions of Known Biologic Function Table 3 Cytokine Protein Abbreviations 132 Table 4 Growth Factor Abbreviations 132 Table 5 Apoptosis Protein Abbreviations 135 viii LIST OF FIGURES Figure 1 Fibronectin Functional Domains 12 Figure 2 The ILK-Kindlin Mechanotransduction Axis 46 Figure 3 The Paxillin-Talin and VASP-Actinin Mechanotransduction Axes 47 Figure 4 Force-Inducible Magnetic Bead Co-Immunoprecipitation Protocol 62 Figure 5 Citrullination is Verified via SDS PAGE, COLDER Assay, and Dot 64 Blot Figure 6 Analysis of PAD Isotype-Specific Fibronectin Citrullination Sites 65 Figure 7 Attribution of Possible Biological Function for Identified Fn 66 Citrullination Sites Figure 8 Bio-Layer Interferometry Average Kon and KD values 67 Figure 9 Alpha 5 Beta 1 Interferometry Curves with Fn 68 Figure 10 Alpha 5 Beta 1 Interferometry Curves with Cit Fn 69 Figure 11 Overlay of Fn and Cit Fn Alpha 5 Beta 1 Interferometry Results 69 Figure 12 Adhesion Assays with Integrin-Specific Binding 70 Figure 13 Integrin ICC Staining and Force-Inducible Co-Immunoprecipitation 72 Assays Display a Preference for Alpha5 and Beta1 on Cit Fn Figure 14 Integrin Expression in Human Lung Fibroblasts 73 Figure 15 ICC Integrin Staining of Beta1-Knockdown Fibroblasts 74 Figure 16 Downstream p-FAK, p-SRC, and p-ILK Signaling 75 Figure 17 Glycogen Synthase Kinase (GSK) Analysis 76 Figure 18 Mechano-sensitive Protein Products