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University of Florida Thesis Or Dissertation Formatting MOLECULAR MECHANISMS OF IRON UPTAKE BY HEPG2 HEPATOMA CELLS By LIN ZHANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012 1 © 2012 LIN ZHANG 2 To my beloved wife: without your endless love and support, none of these would have meanings 3 ACKNOWLEDGMENTS I would like to express my deepest appreciation to my super advisor, Dr. Mitchell Knutson, who gave me the chance to study in the department of Food Science and Human Nutrition. I was very impressed by not only his enthusiasm toward research, but his excellent personality as well. I have been learning from him what I need to know to be a good scientist and a good advisor. Without his persistent help and guidance, this thesis would not have been successful. Additionally, I would like to thank each of my committee members: Dr. James Collins, and Dr. Michael King. They were abundantly helpful and offered invaluable advice, and guidance. Their efforts for my thesis were highly valued and appreciated. I would like also thank all my lab members: Chia-yu Wang, Supak Jenkitkasemwong, Wei Zhang, and Richard Coffey for help. I had a wonderful time with them both in and out of the lab. Their help makes this thesis much easier to accomplish. Finally, I would like to thank my wife--Yan Ren, my parents, and all the family members, for their support and encouragement for me to pursue this degree. Thank you all. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 11 2 LITERATURE REVIEW .......................................................................................... 15 Introduction to Iron .................................................................................................. 15 Iron Metabolism ...................................................................................................... 15 Biological Functions of Iron .............................................................................. 15 Whole Body Iron Distribution ............................................................................ 17 Iron Recycling ................................................................................................... 17 Iron Storage ...................................................................................................... 18 Iron Excretion ................................................................................................... 19 Iron-Related Diseases ...................................................................................... 19 Liver Iron Metabolism.............................................................................................. 20 Function of Liver in Iron Metabolism ................................................................. 20 Hepcidin ..................................................................................................... 21 HFE ............................................................................................................ 22 TF .............................................................................................................. 22 TFR2 .......................................................................................................... 23 Hepatic Iron Uptake ................................................................................................ 24 TBI Uptake ....................................................................................................... 24 TFR1-mediated TBI uptake ........................................................................ 24 TFR2-mediated TBI uptake ........................................................................ 26 TFR1/2-independent TBI uptake ................................................................ 27 NTBI Uptake in the Liver .................................................................................. 28 The Role of Endocytosis in TBI Uptake into Hepatocytes ....................................... 30 Endocytosis-Dependent TBI Uptake ................................................................ 30 TFR1/2-mediated endocytosis ................................................................... 30 Fluid-phase endocytosis (pinocytosis) ....................................................... 32 Endocytosis-Independent Pathway .................................................................. 33 Reductive iron release at the cell surface ......................................................... 34 Iron Transporters in Hepatocytes ............................................................................ 35 DMT1................................................................................................................ 35 ZIP14 and Iron Metabolism .............................................................................. 37 Identification and characterization of ZIP14 ............................................... 37 5 ZIP14 and cellular iron uptake ................................................................... 38 Specific Aims .......................................................................................................... 40 Aim 1. To Test the Hypothesis that Endocytosis Is not Required for TBI Uptake in HepG2 Cells. ................................................................................. 40 Aim 2. To Test the Hypothesis that TF Interacts with ZIP14............................. 40 3 MATERIALS AND METHODS ................................................................................ 41 Knockdown of Clathrin and TFR1/2 Using RNAi ..................................................... 41 Measurement of TF Uptake and Assimilation of Iron from TF ................................. 41 DuoLink In Situ Proximity Ligation Assay ................................................................ 42 Far-Western Blotting Analyses ............................................................................... 43 Co-Immunoprecipitation Assay ............................................................................... 43 Statistical Analysis .................................................................................................. 44 4 RESULTS ............................................................................................................... 45 Inhibiting Clathrin-Mediated Endocytosis in HepG2 cells Does not Affect Iron Uptake from TF .................................................................................................... 45 The Effects of Knocking down TFR1/2 on TF Uptake Are Different at Low and High TF Levels .................................................................................................... 46 ZIP14 Can Interact with TF both In Vitro and In Vivo .............................................. 48 5 DISCUSSION ......................................................................................................... 51 LIST OF REFERENCES ............................................................................................... 55 BIOGRAPHICAL SKETCH ............................................................................................ 69 6 LIST OF FIGURES Figure page 1-1 Assimilation of iron through TFR-dependent pathway in developing erythroid cells .................................................................................................................... 12 4-1 Clathrin heavy chain (CHC) knockdown with siRNA decreases uptake of TF, but does not affect uptake of iron from TF.. ........................................................ 45 4-2 TFR1 and TFR2 knockdown with siRNA decreases uptake of TF at 50 nM level.. .................................................................................................................. 46 4-3 TFR1 and TFR2 knockdown with siRNA increases uptake of TF at 5 µM level.. .................................................................................................................. 47 4-4 ZIP14 can interact with TF in vivo in FLAG-tagged HepG2 cells.. ...................... 48 4-5 ZIP14 can bind TF directly in vitro.. .................................................................... 49 4-6 ZIP14 interact with endogenous TF.. .................................................................. 50 7 LIST OF ABBREVIATIONS AAV Adeno-associated virus BSA Bovine serum albumin CO-IP co-immunoprecipitation DAPI 4', 6-diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMT1 Divalent metal transporter 1 FENTA Iron-nitrilotriacetate FPN1 Ferroportin 1 HFE High Fe gene (Gene mutated in the most common form of hemochromatosis) HH Hereditary hemochromatosis HRP Horseradish peroxidase IRT Iron-regulated transporter IRP Iron-regulatory protein IRE Iron-responsive element KDA Kilodalton LPS Lipopolysaccharide NTBI Non-transferrin-bound iron PFA Paraformaldehyde PLA Proximity Ligation Assay RES Reticuloendothelial system RNAI RNA interference ROS Reactive oxygen species SFM
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