REDOX-SENSITIVE REGULATION OF MRTF-A PHOSPHORYLATION VIA PALLADIN IN VASCULAR SMOOTH MUSCLE CELL DIFFERENTIATION 公开
Lee, Minyoung (2013)
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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