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

et al. (2013) Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/ Myocardin Signaling. PLoS ONE 8(3): e58746. doi:10.1371/journal.pone.0058746 Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling Xuehui Yang 0 1 Yan Gong 0 1 Yuefeng Tang 0 1 Hongfang Li 0 1 Qing He 0 1 Lindsey Gower 0 1 Lucy Liaw 0 1 Robert E. Friesel 0 1 Gianfranco Pintus, University of Sassari, Italy 0 Current address: Program in Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts , United States of America 1 1 Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America, 2 Graduate School for Biomedical Sciences, University of Maine, Orono, Maine, United States of America, 3 Department of Physiology, College of Basic Medicine, Lanzhou University , Lanzhou , China Background: Changes 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 Findings: Here, 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. Conclusions: 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. - Funding: This work was supported by National Institutes of Health grants R01 HL065301 and P30RR030927/P30GM103392 (confocal microscopy, DNA sequencing, and viral vector cores) RF, PI, R01 Hl070865 to LL, and P20RR181789/P20GM103465 (cell phenotyping core) to DM Wojchowski, PI, and institutional support from the Maine Medical Center. YG is the recipient of a predoctoral fellowship award from the Founders Affiliate of the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. VSMC in the adult vessel exhibit a fully differentiated phenotype with low proliferation and high expression of smooth muscle markers including SM alpha-actin (ACTA2), SM22 alpha (SM22a), calponin-h1 (CNN1), SM myosin heavy chain (MYH11), and smoothelin B (SMTN-B) [1,2]. However, VSMC in the intact vessel wall retain phenotypic plasticity, and can respond to environment changes, such as injury, by loss of expression of contractile genes, hyperproliferation, and migration to form a neointima. Elucidation of the mechanisms of VSMC phenotypic switching is critically important to provide insight towards a better understanding of the development of vascular disease and its treatment. Cellular signaling pathways including phosphatidylinositol 3kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (MAPK/ERK) and p38 mitogen-activated protein kinase (p38) regulate VSMC phenotypic modulation [3,4,5,6,7]. The PI3K/Akt signaling pathway promotes both proliferation and, paradoxically, differentiation of VSMC, while MAPK/ERK signaling mediates proliferation and migration of VSMC. The expression of VSMC marker genes is dependent upon a cis-acting DNA sequence, the CArG box, which binds serum response factor (SRF). 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]. Elk1, 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. Phosphorylation of FoxOs by Akt leads to their exclusion from the nucleus and inhibiting their transcriptional functions. When Akt signaling is low in VSMC, nuclear FoxO4 forms a complex with Myocd and inhibits transcription of CArG box-dependent genes [10]. Activation of the Akt pathway in VSMC results in dissociation of FoxO4-Myocd complexes and expression of VSMC marker genes. Spry proteins are feedback regulators of receptor tyrosine kinases (RTKs) that restrain RTK-mediated ERK signaling [11,12], and therefore play critical roles in the regulation of cell proliferation, differentiation, and survival. By inhibiting ERK signaling, Sprys regulate cell proliferation and differentiation in many cell types including endothelial [13,14], C2C12 [15], and skeletal muscle satellite cells [16]. Regulation of Akt activation by Sprys is less well understood. One report showed that human Spry2 expression in HeLa cells increases PTEN expression, decreases its phosphorylation and increases its phosphatase activity, leading to decreased Akt activation [17]. However, potential regulation of Akt by Spry in nontransformed cells has not been addressed. In addition, although a TAT-hSpry2 fusion protein was previously reported to inhibit rat VSMC proliferation and migration in vitro, and injury induced neointima formation in vivo [18], Spry1 and Spry4, which are both highly expressed in normal blood vessels, have not been studied in VSMC. Because of the importance of ERK and Akt signaling in VSMC phenotypic modulation, we addressed the role of Sprys in determining VSMC phenotype. In this study, we report for the first time that Spry1 and Spry4 have opposing effects on Akt activation. Spry1 induces and maintains Akt activation and subsequent expression of VSMC marker genes, while Spry4 inhibits Akt activation and inhibits VSMC proliferation, migration, and VSMC marker gene expression. Using siRNA knockdown and ChIP assays, we demonstrate that FoxO3a binds to the Myocd promoter and negatively regulates My (...truncated)