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Redox Regulation of Estrogen Receptor Alpha and Sodium Hydrogen Exchanger 1 Gene Expression by Hydrogen Peroxide Chua Sui Huay B

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Redox Regulation of Estrogen Receptor Alpha and Sodium Hydrogen Exchanger 1 Gene Expression by Hydrogen Peroxide Chua Sui Huay B REDOX REGULATION OF ESTROGEN RECEPTOR ALPHA AND SODIUM HYDROGEN EXCHANGER 1 GENE EXPRESSION BY HYDROGEN PEROXIDE CHUA SUI HUAY BSc (HONOURS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I want to express my heartfelt gratitude for my supervisor Associate Professor Marie- Veronique Clement, Department of Biochemistry. I am thankful that she allowed me to join the lab during my honors year and for continuing my graduate study under her. A/P Clement was not just a supervisor, but a mentor who is always willing to listen, to encourage and to share her vast knowledge. I would like to thank her for her constant encouragement, patience and faith in my abilities to complete this study. I would also like to thank my co-supervisor Dr Alan Kumar for his guidance and support throughout the course of my graduate study in NUS. I am grateful for Professor Shazib Pervaiz for being on my thesis advisory committee and for his positive feedback on the direction of my project. My warmest thanks to my lab mates in Biochemistry department, for making the long days in lab enjoyable. Special thanks to San Min and Jeya who helped me to proof- read my thesis. I also want to thank Mui Khin for helping me with all the ordering of reagents for my project. Thank you NUMI girls for being my great lab mates and friends. Thanks for being there to cheer me on and also for being part of my sister gang during my wedding. I will certainly miss those days that we hung out together. Finally, my deepest gratitude to my family for their encouragement and support throughout the years, and a special thanks to Zong Hao, who has stood by me through the good and bad times. ii SUMMARY There is mounting evidence that implicates reactive oxygen species (ROS) as an important signaling molecule in various physiological processes. In contrast to the wealth of information on gene up-regulation by ROS, little attention has been paid to the down-regulation of gene expression by ROS. In this thesis, we have studied how two important genes, estrogen receptor alpha (ER α) and sodium hydrogen exchanger 1 (NHE1) were redox-regulated by H 2O2. In the first part of the study, the regulation of ER α by oxidative stress induced by the exposure of MCF7 cells to H 2O2 was investigated. The data supported that ER α protein is down-regulated when exposed to oxidative stress. The down-regulation of ER α protein occurs not through proteosomal degradation pathway, but via the decrease in ER α mRNA level. We found that Akt, MAPK and caspases were not involved in the down-regulation of ER α by H 2O2. Instead, H2O2 inhibition of ER α expression involves an oxidation event that is reversible by the addition of reducing agent, DTT. We also demonstrated that 15d-PGJ2 suppressed ER α expression via the production of ROS. ER α down-regulation resulted in decreased ER α-responsive gene expression and impaired estrogen signaling. These effects could have contributed to the growth arrest observed in H2O2 treated MCF7 cells. In the second part of the study, the down-regulation of NHE1 by H2O2 was examined. We found that the minimal promoter region required for full transcription activation lies on the 0.15kb of NHE1 promoter. This region is also regulated by H2O2. The regulation of NHE1 by H2O2 is similar to the regulation of ER α. H2O2 targets NHE1 at the mRNA level in a reversible manner. The down-regulation involves an oxidation mechanism which is reversible by reducing agents DTT and BME. The drug 15d- iii PGJ2 was also shown to down-regulate NHE1 promoter activity via the production of ROS. Finally, the data suggested that AP2 is not the transcription factor for NHE1 and is not a target in H 2O2 mediated down-regulation of NHE1. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS …………………………………………………..………II SUMMARY………………………………………………………………………….III TABLE OF CONTENTS……………………...……………………………………...V LIST OF TABLES…………………………………………………………………....X LIST OF FIGURES………………………………………………..………………...XI ABBREVIATIONS……………………………………………………………...…XVI PUBLICATIONS AND PRESENTATIONS……………………………………...XIX CHAPTER 1: INTRODUCTION .................................................................................. 1 1.1 FREE RADICALS, REACTIVE SPECIES AND REDOX BALANCE ....... 1 1.1.1 Reactive oxygen species .......................................................................... 1 1.1.2 The antioxidant system ............................................................................ 5 1.1.3 Hydrogen peroxide as a signaling molecule ............................................ 7 1.2 REDOX REGULATION OF GENE EXPRESSION ................................... 10 1.2.1 Transcriptional regulation ...................................................................... 10 1.2.2 mRNA stability ...................................................................................... 13 1.2.3 Direct oxidative modification ................................................................ 14 1.2.4 Regulation of protein turnover ............................................................... 16 1.3 ESTROGEN RECEPTOR ............................................................................ 17 1.3.1 Estrogen and estrogen receptors: Introduction ...................................... 17 1.3.2 Estrogen receptor and genomic signaling .............................................. 20 1.3.3 Membrane and cytoplasmic ER signaling ............................................. 21 1.3.4 Regulation of estrogen receptors ........................................................... 23 1.3.5 Estrogen and estrogen receptors in human breast cancer ...................... 27 v 1.4 THE SODIUM HYDROGEN EXCHANGER 1 .......................................... 29 1.4.1 pH regulation in the cells ....................................................................... 29 1.4.2 Mammalian Na +/H + exchanger .............................................................. 30 1.4.3 NHE1: Basic structure ........................................................................... 31 1.4.4 Physiological and pathological roles of NHE1 ...................................... 33 1.4.5 Regulation of NHE1 .............................................................................. 37 1.4.6 Regulation of NHE1 gene transcription ................................................. 39 1.5 AIM OF STUDY ........................................................................................... 41 CHAPTER 2: MATERIALS AND METHODS ......................................................... 43 2.1 MATERIALS ................................................................................................ 43 2.1.1 Chemicals and reagents.......................................................................... 43 2.1.2 Antibodies .............................................................................................. 44 2.1.3 Plasmids ................................................................................................. 44 2.1.4 Cell lines and cell culture ....................................................................... 45 2.2 METHODS.................................................................................................... 47 2.2.1 Treatment of cells with hydrogen peroxide (H 2O2) and other compounds………………………………………………………………………47 2.2.2 Morphology studies ............................................................................... 47 2.2.3 Crystal violet assay ................................................................................ 47 2.2.4 Plasmid transfection ............................................................................... 48 2.2.5 Small interfering RNA (siRNA) transfection ........................................ 48 2.2.6 Protein concentration determination ...................................................... 49 2.2.7 SDS-PAGE and western blot ................................................................. 50 2.2.8 Caspase assay ......................................................................................... 51 2.2.9 Single and dual luciferase assay ............................................................ 52 2.2.10 Chloramphenicol acetyl transferase (CAT) assay.................................. 53 2.2.11 RNA isolation ........................................................................................ 54 2.2.12 Reverse transcription (RT) and real-time chain polymerase reaction (PCR)……………………………………………………………………………54 2.2.13 Intracellular ROS measurement by CM-H2DCFDA ............................. 55 2.2.14 Nuclear-cytoplasmic fractionation ......................................................... 55 2.2.15 Electromobility shift assay (EMSA) ...................................................... 56 vi 2.2.16 Statistical analysis .................................................................................. 57 CHAPTER 3A: RESULTS – REDOX REGULATION OF ER α ............................... 58 3A.1 H2O2 AND THE EXPRESSION OF ER α PROTEIN................................... 58 3A.1.1 Morphology and growth of MCF7 cells upon exposure to H 2O2 .......... 58 3A.1.2 Effect of H 2O2 on the expression of ER protein .................................... 61 3A.1.3 Effects of estrogen on H 2O2 down-regulation of ER α ........................... 66 3A.1.4 Time-dependent regulation of ER α protein by H 2O2 ............................. 68 3A.1.5 Chronic ROS leads to continuous suppression of ER α .......................... 70
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