Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Xuehui Yang,Qing He,Yuefeng Tang,Yan Gong,Robert E Friesel,Lucy Liaw,Hongfang Li,Lindsey Gower,Gianfranco Pintus

doi:10.1371/journal.pone.0058746

Abstract

BackgroundChanges in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.Methodology/Principal FindingsHere, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.ConclusionsTogether, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.

Highlights

Phenotypic modulation of vascular smooth muscle cells (VSMC) plays a critical role in the development of vascular diseases such as atherosclerosis and post-angioplasty restenosis [1,2]
Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway
The expression of VSMC marker genes is dependent upon a cis-acting DNA sequence, the CArG box, which binds serum response factor (SRF)

Summary

Introduction

Phenotypic modulation of vascular smooth muscle cells (VSMC) plays a critical role in the development of vascular diseases such as atherosclerosis and post-angioplasty restenosis [1,2]. Myocardin (Myocd) is a potent transcriptional co-activator of SRF, forms an SRF/Myocd transcriptional complex and drives CArG box-dependent VSMC marker gene expression [8]. A downstream target of ERK, is a potent repressor of the VSMC contractile gene transcription program by binding to SRF and inhibiting complex formation with Myocd [9]. Phenotypic modulation of VSMC by activation of the Akt pathway occurs in part through FoxO transcription factors. When Akt signaling is low in VSMC, nuclear FoxO4 forms a complex with Myocd and inhibits transcription of CArG box-dependent genes [10]. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established

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