Sphingosine 1-Phosphate Induces Differentiation of Mesoangioblasts towards Smooth Muscle. A Role for GATA6 Chiara Donati1,2*, Giuseppina Marseglia3, Alberto Magi3, Simona Serratı`4, Francesca Cencetti1,2, Caterina Bernacchioni1,2, Genni Nannetti3, Matteo Benelli3, Silvia Brunelli5,6, Francesca Torricelli3, Giulio Cossu5,7, Paola Bruni1,2 1 Dipartimento di Scienze Biochimiche, Universita` di Firenze, Firenze, Italia, 2 Istituto Interuniversitario di Miologia (IIM), Italia, 3 SOD di Diagnostica Genetica, Azienda Ospedaliero Universitaria, Firenze, Italia, 4 Dipartimento di Oncologia e Patologia Sperimentali, Universita` di Firenze, Firenze, Italia, 5 Divisione di Medicina Rigenerativa, Istituto Scientifico H San Raffaele, Milano, Italia, 6 Dipartimento di Medicina Sperimentale, Universita` di Milano-Bicocca, Monza, Italia, 7 Dipartimento di Biologia, Universita` di Milano, Milano, Italia Abstract Different cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFb. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFb abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFb. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration. Citation: Donati C, Marseglia G, Magi A, Serrat`ı S, Cencetti F, et al. (2011) Sphingosine 1-Phosphate Induces Differentiation of Mesoangioblasts towards Smooth Muscle. A Role for GATA6. PLoS ONE 6(5): e20389. doi:10.1371/journal.pone.0020389 Editor: Marcello Rota, Brigham & Women’s Hospital - Harvard Medical School, United States of America Received December 13, 2010; Accepted May 2, 2011; Published May 24, 2011 Copyright: 2011 Donati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from University of Florence (ex-60%) to P. Bruni and C. Donati, www.unifi.it, Ente Cassa di Risparmio di Firenze to F. Torricelli, www.crf.it, Fondazione Cassa di Risparmio di Lucca to P.Bruni, www.fondazionecarilucca.it. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chiara.donati@unifi Introduction Smooth muscle (SM) cells control many fundamental functions such as arterial tone and airway resistance; alterations in vascular SM cells contribute to a number of diseases in humans including atherosclerosis and hypertension [1]. SM cells are not terminally differentiated and are able to switch between a contractile and synthetic phenotype in response to changing local environmental cues [2]. A large number of factors including mechanical forces, extracellular matrix components, endothelium-SM interactions and transforming growth factor-b (TGFb) have been shown to promote SM marker gene expression in cultured cell systems [1]. However, in contrast to skeletal muscle development, no master genes have been found to regulate smooth muscle development, although several genes such as myocardin, MRTFA, MRTFB, Necdin and Msx2 have been found to be involved in the process [3,4]. Several recent studies demonstrated that circulating, SM progenitor cells can contribute to neointima formation and repair following vascular injury [5]. Mesoangioblasts are a new type of progenitor cells, isolated from explants of dorsal aorta, capable of differentiating into various mesoderm cell types, such as smooth and striated muscle, bone and endothelium [6]. When delivered in the left ventricle, mesoangioblasts cause significant functional recovery despite modest anatomical repair of infarcted cardiac muscle [7]. The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a lipid mediator that regulates fundamental biological processes mainly through binding to its specific receptors S1P1–5 in many cell systems [8]. S1P has recently been shown to have interesting effects on vascular development and SM cells growth and migration. S1P stimulates angiogenesis and induces vascular maturation in many experimental models [9]. Recent literature highlights the role of S1P in the regulation of proliferation, survival, differentiation and migration of a range of adult and embryonic stem cells [10]. We previously demonstrated that S1P acts as potent mitogen and anti-apoptotic agent in murine and human mesoangioblasts [11]. The important role of S1P in these cells, is further supported by our finding that the anti-apoptotic PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20389