Abstract
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
TABLE OF CONTENTS
Page #
ACKNOWLEDGEMENTS.....................................................................................vi
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
1.1.1.1
Signaling 5
1.1.1.2
Gene Expression
7
1.1.1.3
Proliferation and Growth
9
1.1.1.4
Differentiation
11
1.1.1.5
Cytoskeletal Remodeling and
Migration 12
1.1.1.5.1
Extension of the
Plasma Membrane at the Cell's Leading Edge 13
1.1.1.5.2
Formation of Focal
Complexes and Adhesions for Cell Movement 16
1.1.1.5.3
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
1.2.2.1
Nox1 20
1.2.2.2
Nox3 20
1.2.2.3
Nox4 21
1.2.2.4
Nox5 24
1.2.3
Classical Nox
Regulatory Proteins
25
1.2.3.1
p22phox 25
1.2.3.2
p47phox and NoxO1
26
1.2.3.3
p67phox and NoxA1
27
1.2.3.4
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
2.3.1.1
Yeast Two-Hybrid
44
2.3.1.2
Association of p22phox and
Poldip2 49
2.3.1.3
Poldip2 Co-localizes with
p22phox in Vascular Smooth Muscle
Cells 52
2.3.2
Poldip2 Association
with Other NADPH Oxidase Subunits
56
2.3.2.1
Association of Poldip2 with
Nox4 and Nox1 in Vascular
Smooth Muscle Cells 56
2.3.2.2
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
3.3.2.1
The Increase in ROS
Production by Poldip2 Occurs via Nox4
and in a p22phox-Dependent Manner 81
3.3.2.2
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
3.3.3.1
Characterization of siPoldip2
90
3.3.3.2
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
About this Dissertation
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