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EHD1 As a Positive Regulator of Macrophage Colony-Stimulating Factor-1 Receptor

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EHD1 As a Positive Regulator of Macrophage Colony-Stimulating Factor-1 Receptor University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Summer 8-19-2016 EHD1 As a Positive Regulator of Macrophage Colony-Stimulating Factor-1 Receptor Luke R. Cypher University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Allergy and Immunology Commons, Infectious Disease Commons, Medical Biochemistry Commons, and the Medical Cell Biology Commons Recommended Citation Cypher, Luke R., "EHD1 As a Positive Regulator of Macrophage Colony-Stimulating Factor-1 Receptor" (2016). Theses & Dissertations. 119. https://digitalcommons.unmc.edu/etd/119 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. EHD1 AS A POSITIVE REGULATOR OF MACROPHAGE COLONY-STIMULATING FACTOR-1 RECEPTOR By Luke Cypher A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Cancer Research Graduate Program Under the supervision of Professor Hamid Band University of Nebraska Medical Center Omaha, Nebraska June 23rd, 2016 Supervisory Committee Members Tammy Kielian, Ph.D. Joyce Solheim, Ph.D. Jing Wang, Ph.D. I would like to dedicate this dissertation to my mother, Kimberly A. Cypher, Ph.D. EHD1 AS A POSITIVE REGULATOR OF MACROPHAGE COLONY-STIMULATING FACTOR-1 RECEPTOR Luke R. Cypher, Ph.D. University of Nebraska, 2016 Supervisor: Hamid Band, M.D., Ph.D. The master regulator of the macrophage development, differentiation, proliferation, survival, phagocytosis, cytokine secretion, motility, adhesion, migration, and spreading is the receptor tyrosine kinase (RTK) colony stimulating factor-1 receptor (CSF-1R). Aberrant CSF-1R signaling is present amongst a variety of highly prevalent and devastating human diseases in the United States such as atherosclerosis, cancer, inflammatory bowel disease, arthritis, and neuro-demyelination/neuro-degeneration. A better understanding of basic mechanisms that govern macrophage development and function is of vital importance in treating patients afflicted with these conditions/diseases. CSF-1R presentation on the macrophage cell surface is a required precursor for CSF1- induced RTK dimerization (activation) and downstream CSF-1R signaling cascades. Mechanisms which regulate CSF-1R trafficking are unstudied and mechanisms of RTK trafficking regulation are poorly understood. The evolutionarily conserved C-terminal Eps15 Homology Domain (EHD) protein family consists of vesicular trafficking regulating proteins. However, the role of EHD proteins in CSF-1R trafficking and signaling has not been studied. I have utilized primary (non-immortalized) murine/mammalian macrophages under inducible Ehd1 gene deletion/knockout (EHD1-KO) to explore the role of EHD1 in CSF-1R trafficking in macrophages. I have discovered an entirely novel function for EHD1 in anterograde transport and presentation of CSF-1R on the macrophage cell surface. EHD1-KO macrophages have significantly depleted total and surface CSF-1R expression (i.e. receptor available for ligand-binding and subsequently CSF-1R activation/signaling) when compared with control macrophages. In EHD1-KO macrophages, newly synthesized CSF-1R en route to the cell surface is essentially shunted to the lysosome and degraded. These findings reveal an entirely novel and essential role for EHD1 in anterograde transport/presentation of CSF-1R to the macrophage cell surface. I ACKNOWLEDGEMENTS I sincerely thank Dr. Hamid Band for his foresight, guidance, understanding, and patience throughout the course of my training to become a physician scientist. Thank you for believing me and providing me with all the tools and resources needed for my thesis project. I thank my graduate committee members: Jenny Wang Ph.D. and Tammy Kielian Ph.D. for all of their advice and evaluation during my time as a Ph.D. student and Joyce Solheim Ph.D. whose door was always open. To Richard Stanley, my Ph.D. would have been impossible without your detailed and transparent protocols. Your method’s sections taught me macrophage cell culture. Thank You To Adam Hoppe’s laboratory from South Dakota State University (SDSU). It has been a joy to work with your lab. I greatly appreciate your flexibility and willingness to work with me on macrophage/CSF-1R imaging. The insight, knowledge, and technical savvy your lab brought to my project was invaluable, and I thank you for it. To all the current and former members of the Hamid Band laboratory. I will always remember the times we shared and friendships formed—Cheers! II TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................................................................... I TABLE OF CONTENTS ....................................................................................... II LIST OF FIGURES ............................................................................................... V LIST OF TABLES .............................................................................................. VIII LIST OF ABBREVIATIONS ................................................................................. IX CHAPTER 1: INTRODUCTION ............................................................................ 1 The monocyte/macrophage cell lineage .................................................... 2 CSF-1R as the master regulator of macrophages ..................................... 4 C-terminal Eps15 Homology Domain (EHD) containing proteins ............... 6 Mouse models to study EHD proteins ........................................................ 7 Hypothesis ................................................................................................. 9 CHAPTER 2: MATERIALS & METHODS ........................................................... 28 Materials .................................................................................................. 29 Mouse Models ......................................................................................... 30 Bone Marrow-Derived Macrophages (BMDMs) ....................................... 31 Fluorescence Activated Cell Sorting (FACS) ........................................... 31 Quantitative Real-Time-PCR (qRT-PCR) ................................................. 32 Immunoblotting (IB) ................................................................................. 32 Immunofluorescence (IF) ......................................................................... 33 3H-thymidine incorporation assay of cellular proliferation ........................ 34 CFSE dye dilution assays to access macrophage proliferation ............... 34 III Macrophage CSF1-induced spreading assay .......................................... 35 Macrophage CSF1-induced migration assay ........................................... 35 [35S]-methionine/cysteine metabolic labeling of CSF-1R ......................... 35 Immunoprecipitation (IP) .......................................................................... 36 Statistics .................................................................................................. 36 CHAPTER 3: RESULTS ..................................................................................... 52 Expression of EHD proteins in Ehd1-WT and Ehd1-null BMDMs ............ 53 Deletion of EHD1 in BMDMs leads to reduced CSF1-induced responses53 Deletion of EHD1 in BMDMs leads to reduced CSF-1R signaling ........... 54 Reduced surface CSF-1R expression in EHD1-KO BMDMs ................... 55 EHD1 inducible deletion in BMDMs ......................................................... 55 Inducible deletion of EHD1 in BMBMs from Ehd1fl/ fl; CreERT2 mice. ......... 56 Tamoxifen is not toxic to BMDMs in vitro ................................................. 57 Inducible EHD1-KO results in reduced CSF1-induced macrophage functions .................................................................................................. 58 EHD1-KO BMDMs have reduced CSF-1R signaling ............................... 59 CSF-1R internalization and degradation are similar in EHD1-KO BMDMs ................................................................................................................. 60 Reduced surface CSF-1R expression in EHD1-KO BMDMs ................... 60 EHD1 deletion results in depletion of CSF-1R protein in BMDMs ............ 61 EHD1 functions to transport newly synthesized CSF-1R to the cell surface ................................................................................................................. 61 CSF-1R was found localizes to this EHD1+/GM130+ compartment ......... 63 IV Newly synthesized CSF-1R transits to lysosomes for degradation in EHD1-KO BMDMs ................................................................................... 64 CHAPTER 4: DISCUSSION & CONCLUSION ................................................. 125 Discussion ............................................................................................. 126 Conclusion ............................................................................................. 133 BIBLIOGRAPHY ............................................................................................... 138 V LIST OF FIGURES Figure 1.1. Macrophages in human disease. 16 Figure 1.2. The origins
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