Lyle, Alicia Nicole (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/k643b168d?locale=en


Reactive oxygen species (ROS), such as superoxide (O2 •-) and hydrogen peroxide (H2O2), are implicated in the development of cardiovascular disease pathologies, including atherosclerosis and restenosis. Physiologically, ROS mediate functions including proliferation, gene expression, migration, differentiation, and cytoskeletal remodeling. One major source of ROS is the NADPH oxidase (Nox) enzymes. In vascular smooth muscle cells (VSMCs), the regulatory proteins that associate with individual Nox homologues are poorly defined. The membrane-bound Nox subunit heterodimerizes with p22phox to form the catalytic moiety and p22phox serves as the docking site for regulatory subunits. Using the cytosolic c-terminal tail of p22phox for a yeast two-hybrid screen, we identified Poldip2, polymerase delta interacting protein 2, as a novel p22phox binding partner.

Immunoprecipitation and co-localization experiments confirm the association of Poldip2 with p22phox. Poldip2 functionally associates with the Nox4/p22phox complex in a p22phox-dependent manner and significantly increases Nox4 enzymatic activity and Nox4-dependent ROS production, thus establishing Poldip2 as a novel positive modulator of Nox4. Furthermore, functional studies indicate that Poldip2 may negatively modulate Nox1 enzymatic activity. In VSMCs, Nox1 and Nox4 are differentially regulated by agonists and exhibit distinct subcellular localization patterns. The data presented establish that Poldip2 is required for proper localization and trafficking of the Nox4/p22phox complex to focal adhesions. Activation of Nox4/p22phox by Poldip2 promotes ROS-dependent activation of RhoA, strengthens focal adhesions and increases stress fiber formation, while depletion of either Poldip2 or Nox4 results in a loss of these structures. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess or insufficient Poldip2, thereby implicating Nox4/p22phox/Poldip2 in Rho-dependent cytoskeletal reorganization, focal adhesion turnover, and migration. Additionally, Poldip2 overexpression increases VSMC polyploidy by blocking cell cycle progression through G2/M.

This is the first report of a protein that functions to positively regulate Nox4 activity and Nox4-dependent ROS production. These data altogether link ROS production by Nox4/Poldip2 to the regulation of cellular functions dependent on tight coordination of cytoskeletal regulation, such as migration and cell cycle progression. Therefore, Poldip2 may serve as a novel therapeutic target for vascular pathologies with a VSMC migratory and/or proliferative component, such as restenosis and atherosclerosis.

Table of Contents

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TABLE OF CONTENTS.......................................................................................vii
INDEX OF FIGURES...........................................................................................xiii
LIST OF SYMBOLS AND ABBREVIATIONS......................................................xix

CHAPTER 1: Introduction 1 1.1 Reactive Oxygen Species 2 1.1.1 Physiological Functions of ROS in the Vasculature 5 Signaling 5 Gene Expression 7 Proliferation and Growth 9 Differentiation 11 Cytoskeletal Remodeling and Migration 12 Extension of the Plasma Membrane at the Cell's Leading Edge 13 Formation of Focal Complexes and Adhesions for Cell Movement 16 Generation of Force and Release of Rear Adhesions for Forward Cell Progression 17 1.2 The NADPH Oxidase Enzyme Family 18 1.2.1 Nox2, the Classical Neutrophil NADPH Oxidase 18 1.2.2 The Nox Family Members 19 Nox1 20 Nox3 20 Nox4 21 Nox5 24 1.2.3 Classical Nox Regulatory Proteins 25 p22phox 25 p47phox and NoxO1 26 p67phox and NoxA1 27 Small Molecular Weight G-Protein, Rac 28 1.2.4 Vascular Smooth Muscle NADPH Oxidases 28 1.3 NADPH Oxidases and Cytoskeletal Dynamics 31 1.3.1 Trafficking of NADPH Oxidases 31 1.3.2 Nox Proteins and Focal Adhesion Dynamics 33 1.3.3 Cell Cycle Regulation by ROS in Vascular Smooth Muscle Cells 34 1.4 Objectives of This Dissertation 35 CHAPTER 2: Identification of Poldip2, a Novel Binding Partner for p22phox in Vascular Smooth Muscle Cells 37 2.1 Introduction 38 2.2 Methods 38 2.3 Experimental Results 44 2.3.1 Identification of Poldip2 as a Novel p22phox-Interacting Partner 44 Yeast Two-Hybrid 44 Association of p22phox and Poldip2 49 Poldip2 Co-localizes with p22phox in Vascular Smooth Muscle Cells 52 2.3.2 Poldip2 Association with Other NADPH Oxidase Subunits 56 Association of Poldip2 with Nox4 and Nox1 in Vascular Smooth Muscle Cells 56 Poldip2 Co-localizes with Nox4 in Vascular Smooth Muscle Cells 61 2.3.3 Expression and Tissue Distribution of Poldip2 65 2.4 Discussion 65 CHAPTER 3: Poldip2 Functions as a Regulator of NADPH Oxidase Enzymatic Activity 73 3.1 Introduction 74 3.2 Methods 74 3.3 Experimental Results 80 3.3.1 Poldip2 Overexpression Increases Basal Oxidase Activity in Vascular Smooth Muscle Cells 80 3.3.2 Poldip2 Stimulates ROS Production by Nox4, but Not by Nox1 81 The Increase in ROS Production by Poldip2 Occurs via Nox4 and in a p22phox-Dependent Manner 81 Poldip2 Does Not Stimulate Nox1-Dependent ROS Production in Vascular Smooth Muscle Cells 86 3.3.3 siPoldip2 Significantly Decreases Poldip2 Levels, Decreases Basal NADPH Oxidase Activity, and Changes Cell Phenotype 90 Characterization of siPoldip2 90 Knockdown of Poldip2 Decreases Basal NADPH Oxidase Activity in Vascular Smooth Muscle Cells and Alters Cell Phenotype 94 3.4 Discussion 94 CHAPTER 4: Poldip2 Regulates Proper Nox4 and p22phox Localization, Focal Adhesion Integrity and Stress Fiber Formation 105 4.1. Introduction 106 4.2. Methods 107 4.3. Experimental Results 112 4.3.1. Modulation of Poldip2 or Nox4 Regulates Focal Adhesion and Stress Fiber Formation 112 4.3.2. Poldip2 Regulates Focal Adhesion and Stress Fiber Formation by Activating RhoA via Nox4 114 4.3.3. Modulation of Poldip2 or Nox4 Inhibits Vascular Smooth Muscle Cell Migration 126 4.4. Discussion 136 CHAPTER 5: Poldip2 Controls Vascular Smooth Muscle Cell Ploidy through Modulation of Cytoskeletal Integrity 141 5.1. Introduction 142 5.2. Methods 142 5.3. Experimental Results 145 5.3.1. Overexpression of Poldip2 Increases Frequency of Polyploidy in VSMCs 145 5.3.2. Poldip2 Regulates Cell Cycle Progression 145 5.4. Discussion 151 CHAPTER 6: Poldip2 Influences the Proper Trafficking of Nox4 and p22phox to Focal Adhesions 153 6.1. Introduction 154 6.2. Methods 154 6.3. Experimental Results 157 6.3.1. Poldip2 is Required for Proper Nox4 and p22phox Localization and Trafficking 157 6.3.2. Impaired Trafficking of Nox4/p22phox Results in Increased Endoplasmic Reticulum Stress in Vascular Smooth Muscle Cells 162 6.4. Discussion 164 CHAPTER 7: Discussion 167 7.1. Poldip2, a Novel Binding Partner for p22phox 168 7.1.1. To What Specific Regions of p22phox does Poldip2 Bind? 170 7.1.2. Does Poldip2 Regulate Nox4 and Nox1 Enzymatic Activity? 171 7.1.3. Does Poldip2 Function Like a Classical Oxidase Regulatory Subunit? 174 7.1.4. How does Poldip2 Regulate the Localization and Trafficking of Nox4? 175 7.2. Nox4/Poldip2, Regulators of Cytoskeletal Integrity in Vascular Smooth Muscle Cells 180 7.2.1. How does Nox4/Poldip2 Function to Regulate RhoA? 180 7.2.2. How does Nox4/Poldip2 Function to Regulate Stress Fibers? 182 7.2.3. How does Nox4/Poldip2 Function to Regulate Microtubule Dynamics? 183 7.2.4. What are the Functional Consequences of Improper Cytoskeletal Regulation? 185 7.3. Implications for Nox4/Poldip2 in Cardiovascular Disease Pathologies and Future Directions 187 References 193

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