Abstract
Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-β (TGF-β) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle α-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.
Highlights
In the vasculature, differentiated vascular smooth muscle cells (VSMCs) are critical for physiological homeostasis; strategies to prevent VSMC de-differentiation are attractive targets for pharmacological intervention
While hydrogen peroxide is produced by multiple enzymatic pathways, hydrogen peroxide used in growth- and differentiation-related signaling in aortic VSMCs is derived from NADPH oxidases, Nox1 and NADPH oxidase 4 (Nox4), respectively [2]
We first investigated the effect of transforming growth factor-β (TGF-β) on myocardin-related transcription factor-A (MRTF-A) phosphorylation in human aortic VSMCs
Summary
In the vasculature, differentiated vascular smooth muscle cells (VSMCs) are critical for physiological homeostasis; strategies to prevent VSMC de-differentiation are attractive targets for pharmacological intervention. Nox and Palladin Regulation of MRTF-A Phosphorylation proteins including smooth muscle α-actin (SMA) and calponin (CNN) [1]. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are implicated in the regulation of signaling pathways involved in VSMC growth, differentiation, migration, and inflammation [2]. TGF- β is a major differentiation factor for smooth muscle [3]. Our previous work has shown that knockdown of Nox reduces TGF-β-induced SMA and CNN mRNA and protein expression in VSMCs [4, 5]. Because Nox has been found in the nucleus [6], and Nox regulates SMA transcription[5], a role for Nox in regulation of the transcription factors associated with differentiation marker gene expression is likely
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