Coronin 1B Regulates Platelet-derived Growth Factor-induced Migration and Reactive Oxygen Species Production 公开

Williams, Holly Colette (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/5t34sk12z?locale=zh
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Abstract

Coronin 1B Regulates Platelet-derived Growth Factor-induced Migration and Reactive Oxygen Species Production
By
Holly C. Williams

Platelet derived growth factor (PDGF) plays a pivotal role in cardiovascular disease progression, partially by initiating vascular smooth muscle cell (VSMC) migration Lamellipodia formation is the first step in migration and the creation of this actin rich protrusion is under the tight control of actin polymerizing proteins such as the Arp2/3 complex and actin depolymerizing proteins such as cofilin. Studies show that the actin binding proteins known as coronins regulate actin polymerization via binding to and inhibiting the ARP2/3 complex. Coronins are known to regulate various actin dependent cellular processes including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined. Therefore, the goal of this dissertation was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration.

Coronin 1B (Coro1B) and 1C (Coro1C) are both expressed in VSMCs at the mRNA and protein levels. Downregulation of Coro1B by siRNA increases PDGF-induced migration, while downregulation of Coro1C has no effect. Through kymograph analysis, it was confirmed that Coro1B-mediated increases in migration are directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. Additionally, PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the Arp2/3 complex. VSMCs transfected with a phospho-deficient S2A-Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse, Coro1B phosphorylation is increased in response to vessel injury in vivo. We also found that the Coro1B phosphorylation state is redox sensitive and dephosphorylation of Coro1B is dependent on an okadaic acid sensitive phosphatase. Furthermore, the knockdown of Coro1B increases PDGF-induced NADPH oxidase-derived ROS production, thereby providing a new avenue by which Coro1B can regulate VSMC migration. Our data support the concept that Coro1B is an important participant in PDGF-induced VSMC migration, a critical step in vascular lesion formation.

Table of Contents

Table of Contents

ACKNOWLEDGEMENTS vi

TABLE OF CONTENTS vii

INDEX OF FIGURES xii

INDEX OF TABLES xvi

LIST OF SYMBOLS AND ABBREVIATIONS xvii


Chapter 1: Introduction

1.1 Coronary Heart Disease: Causes, Common Treatments and Complications 2

1.1.1 Atherosclerosis 2
1.1.2 Percutaneous Coronary Intervention 5
1.1.3 Restenosis After Percutaneous Coronary Intervention 7

1.2 Platelet-derived Growth Factor Expression and Signaling 8

1.2.1 Platelet-derived Growth Factor 9
1.2.2 Platelet-derived Growth Factor Receptor Signaling 10

1.3 Reactive Oxygen Species and NADPH Oxidase Signaling 13

1.3.1 Reactive Oxygen Species Signaling 14
1.3.2 NADPH Oxidase Activity and Signaling 15

1.4 Directed Cell Migration 17

1.4.1 Rho GTPase Regulation 18

1.4.2 Rac Regulation of Actin Polymerization and Lamellipodia Formation 18
1.4.3 Regulation of the ARP 2/3 complex 20

1.5 The Coronin Family of Actin Binding Proteins 23

1.5.1 Structure of Type I Coronins 25

1.5.1.1 N-Terminal Domain: N-Terminal Motif and β- Propeller Region 25
1.5.1.2 Middle Domain: C-Terminal Extension and Unique Region 27
1.5.1.3 C- Terminal Domain: Coiled-Coil Domain 28

1.5.2 Function and Regulation of Type I Coronins 28

1.5.2.1 Coronin 1A 29
1.5.2.2 Coronin 1B 30
1.5.2.3 Coronin 1C 32

1.6 Protein Kinase C 33

1.6.1 PKC Structure, Regulation and Activation 34
1.6.2 Expression and Function of PKCs in VSMCs 35

1.7 Objective of Dissertation 36

Chapter 2: Role of Coronin 1B in PDGF-induced VSMC Migration

2.1 Introduction 38
2.2 Materials and Methods 39
2.3 Results 47

2.3.1 Expression of Type I Coronins in VSMC Migration 47
2.3.2 Coronin 1B Down Regulation Potentiates PDGF-induced VSMC Migration 49
2.3.3 Coronin 1B Over Expression Inhibits PDGF-induced Migration 56

2.3.4 Coronin 1B Localizes to the Cytosol and Cell Periphery in VSMCs 59
2.3.5 Coronin 1B Down Regulation Modifies PDGF-induced Changes in Lamellipodia Dynamics 62
2.3.6 PDGF Stimulation of VSMCs Induces Coronin 1B Serine 2 Phosphorylation 66
2.3.7 PDGF-stimulates Coro1B Serine Phosphorylation via PKCε 68
2.3.8 Coronin 1C is Not Phosphorylated by PKC in Response to PDGF in VSMCs 72
2.3.9 Phosphorylation Deficient Coro1B Mutant Decreases VSMC Migration 76
2.3.10 Basal and PDGF-induced Changes in Coronin Protein Interactions 81
2.3.11 PDGF Stimulation Disrupts Coronin1B and ARP2/3 Complex Interaction 84
2.3.12 Coronin 1B is Phosphorylated in the Neointima After Mouse Carotid Artery and Rat Carotid Balloon Injury 84

2.4 Discussion 90

Chapter 3: Coronin 1B and Redox Signaling

3.1 Introduction 96
3.2 Materials and Methods 97
3.3 Results 100

3.3.1 Hydrogen Peroxide Does Not Significantly Increase Coronin 1B Phosphorylation 100
3.3.2 PDGF-induced Coronin 1B Serine-2 Phosphorylation is Augmented by the Deletion of Nox1 100

3.3.3 Mechanism of Coronin 1B Serine-2 Dephosphorylation 102
3.3.4 siCoro1B Increases PDGF-induced NOX-derived ROS 106

3.4 Discussion 108

Chapter 4: Discussion

4.1 VSMC Coronins 112

4.1.1 Coronin 1B Functions and Localization 114

4.1.1.1 Coronin 1B Regulation of the Arp2/3 complex and SSH1L 114
4.1.1.2 Coronin 1B Regulation of ROCK 115
4.1.1.3 Coronin 1B Localization Suggests Other Possible Functions 118

4.1.2 Coronin 1C Functions and Localization 120
4.1.3 Coronin 1B and 1C Complex Formation 122

4.2. Consequences of Coronin Phosphorylation 122

4.2.1 Coronin 1B 122

4.2.1.1 Coronin 1B Serine 2 Phosphorylation 122
4.2.1.2 Predicted Coronin 1B Phosphorylation Sites 124
4.2.1.3 Coronin 1B Dephosphorylation 127

4.2.2 Coronin 1C 128

4.3 Coronin as an Effector of PKC 130
4.4 Coronin and ROS 131
4.5 Coronin and Disease 134

4.5.1 Coronins and Cardiovascular Disease 134
4.5.2 Coronins and Cancer 135
4.5.3 Coronins and the Immune Response 137

4.5.4 Coronins, Neuronal Plasticity, and Schizophrenia 138

4.6 Summary and Future Directions 139

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