NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor

Abstract

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β. KeywordsVascular smooth muscle; smooth muscle α-actin; transforming growth factor-β; NADPH oxidase 4; serum response factor; reactive oxygen species; p38 mitogen activated protein kinase Vascular smooth muscle cells (VSMC) from adult tissue exhibit a differentiated phenotype, which is defined by the expression of contractile proteins such as smooth muscle alpha actin (SMA), calponin (CNN) and smooth muscle myosin heavy chain (SM-MHC) [1]. In contrast to other cell types, VSMC can modify their phenotype in response to external signals, transforming into a synthetic phenotype that is able to proliferate and migrate and secrete increased amounts of matrix proteins. This modification occurs in different cardiovascular diseases such as atherosclerosis, restenosis or hypertension, contributing to their pathophysiology [1]. Redifferentiation of synthetic VSMC limits lesion formation; however, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [5,6]. In addition, Nox4 and its activator Poldip2, appear to regulate stress fiber formation, conceivably by activating RhoA [7]. However, almost nothing is known about the mechanisms by which reactive oxygen species ...

Mitogen-Activated Protein Kinase 14 Is a Novel Negative Regulatory Switch for the Vascular Smooth Muscle Cell Contractile Gene Program

“…44,46,49 These studies suggest that under conditions of TGFβ1 stimulation, p38MAPK signaling is important for VSMC differentiation ( Figure 7). The current study predicted that knockdown of p38MAPK would replicate results obtained with SB203580.…”

“…[39][40][41][42][43][44][45][46][47][48][49][50] p38 mitogen-activated protein kinase (p38MAPK) is among the signaling pathways shown to be pivotal for TGFβ1-induced VSMC differentiation. 44,46,49,51 p38MAPK comprises 4 different isoforms encoded in 4 distinct gene loci. Among these isoforms, p38MAPKα (MAPK14) and p38MAPKβ (MAPK11) have received most attention in terms of expression and activity, based on their high sensitivity to the selective inhibitors, SB203580 and SB202190.…”

“…44,49,51 MKK6 is an upstream, dual-specificity kinase that selectively phosphorylates MAPK14. 52 Overexpressing constitutively active MKK6 in HCASMCs results in a dose-dependent increase in phospho-MAPK14 ( Figure 5A).…”

Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate

“…Activation of p38 MAPK is required for the expression of genes encoding SMC contractile markers and for inhibiting VSMC cell cycle progression. [9][10][11] Recent studies have reported that p38 MAPK is involved in SRF and myocardin expression. During cardiovascular development and VSMC phenotypic modulation, p38 MAPK plays a key role in inducing myocardin expression by activating myocyte enhancer factor 2, an upstream transcription factor of myocardin.…”

“…8 Because the activation of the p38 MAPK pathway is involved in phenotypic switching and cell cycle arrest of VSMCs, we investigated whether p38 MAPK is required for FGF12-induced phenotypic changes in VSMCs. [9][10][11] Western blotting showed that phosphorylation of p38 MAPK was profoundly enhanced in adenovirus FGF12-transfected HASMCs compared with control cells (Figure 7A). Inhibition of the p38 MAPK pathway blocked FGF12-induced upregulation of myocardin and SRF …”