REDOX-SENSITIVE REGULATION OF MRTF-A PHOSPHORYLATION VIA PALLADIN IN VASCULAR SMOOTH MUSCLE CELL DIFFERENTIATION Open Access

Abstract

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in cardiovascular diseases such as atherosclerosis and restenosis. Differentiated VSMCs have robust stress fibers and express contractile proteins, such as smooth muscle α-actin (SMA), necessary for vascular function. Loss of the differentiated phenotype has been observed in disease processes. Understanding the mechanisms involved in these changes is important in understanding and treating these diseases. Our previous studies indicate that transforming growth factor β-1 (TGF-β) induces reactive oxygen species (ROS) through NADPH oxidase 4 (Nox4), which modulate activity of myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF), transcription factors that regulate expression of prodifferentiation genes such as SMA. Regulation of these factors is incompletely understood, but evidence exists for an interaction of MRTF-A with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear. In this thesis, we tested the hypothesis that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SRF responsive genes in VSMCs and that this phosphorylation is dependent upon Nox4-mediated palladin expression and interaction with phosphatase PP2A. Knockdown of Nox4 using siRNA decreases TGF-β-induced palladin expression and MRTF-A phosphorylation suggesting redox-sensitive regulation of the proteins. The phosphorylation of MRTF-A is mediated by ROCK and inhibition of this kinase resulted in a decrease in SMA expression. Palladin also regulates this phosphorylation, because knockdown of palladin decreases MRTF-A phosphorylation. Co- immunoprecipitation assay showed palladin binds to protein phosphatase 2A (PP2A), suggesting the idea that palladin may sequester PP2A from MRTF-A, which results in prevention of MRTF-A dephosphorylation. These data suggest that ROCK-mediated phosphorylation of MRTF-A is important in regulation of SRF responsive gene expression. Furthermore, the data support the idea that the MRTF-A phosphorylation is controlled by Nox4-mediated palladin expression and interaction with PP2A. Therefore, the novel role of Nox4 and palladin in phosphorylation of MRTF-A and subsequent differentiation gene expression in VSMCs may offer a new therapeutic target for atherosclerosis and restenosis.

Table of Contents

Page #

CHAPTER I: Introduction

1

1.1

Diseases: Atherosclerosis, In-stent restenosis

2

1.2

Reactive Oxygen Species and NADPH Oxidase Signaling

3

1.3

TGF-β-mediated Smooth Muscle Cell Differentiation Signaling

6

1.4

MRTF-A and SRF

7

1.5

Palladin

10

1.6

Overall Hypothesis

12

CHAPTER 2: Role of Palladin, Nox4 and MRTF-A in Vascular Smooth Muscle Cell Differentiation Gene Expression

15

2.1

Introduction

16

2.2

Methods

17

2.3

Experimental Results

20

2.3.1

TGF-β-induces Palladin and SMA Expression

20

2.3.2

TGF-β-induced SMA Expression Requires Palladin

21

2.3.3

Induction of Palladin by TGF-β is Redox-Sensitive

21

2.3.4

siNOX4 Blocks Palladin and SMA Expression

21

2.3.5

TGF-β-induced MRTF-A Phosphorylation is Abolished by siPalladin

27

2.3.6

TGF-β-induced MRTF-A Phosphorylation is Redox-Sensitive

31

2.3.7

siNOX4 Blocks MRTF-A Phosphorylation and its Expression

31

2.3.8

TGF-β-induced MRTF-A Phosphorylation is Mediated by ROCK

31

2.3.9

Palladin Does Not Affect Nuclear Localization of MRTF-A

32

2.3.10

Palladin May Sequester PP2A from MRTF-A by Binding to PP2A

37

2.4

Discussion

39

CHAPTER 3: Discussion

43

About this Master's Thesis

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Pharmacology Biology, Molecular
Keyword	Palladin MRTF-A Smooth muscle cell differentiation
Committee Chair / Thesis Advisor	Griendling, Kathy, Emory University
Committee Members	Davis, Michael E, Emory University Sutliff, Roy, Emory University Brown, Lou Ann, Emory University

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