University of Florida Thesis Or Dissertation Formatting

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

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    69 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us