S1P Differentiates Mesoangioblasts Figure 3. Dose-dependence of GATA6 expression level induced by S1P (A) and role of the transcription factors GATA6, LMCD1 (B,C) on the expression levels of SM markers induced by S1P in mesoangioblasts. Mutual regulation of the transcription factors GATA6, LMCD1 and TRPS1 (D,E,F). Real time PCR analysis of GATA6 (A) was performed in cells challenged with the indicated concentrations of S1P for 6 h. Real-Time PCR analysis of CNN1 (B), TPM1 (C), GATA6 (D), LMCD1 (E), TRPS1 (F) was performed in cells transfected with scrambled-, GATA6- or LMCD1- siRNA and stimulated with S1P for 24 h. doi:10.1371/journal.pone.0020389.g003 observed effect. Indeed, incubation of the 3D matrigel culture of mesoangioblasts in the presence of 10 mmol/L SKI-2, significantly impaired the appearance of the differentiated phenotype (Fig. 6B). Although SphK plays a crucial role in the survival of mesoangio- blasts [12], the presence of serum in the growing medium prevented the onset of the apoptotic cell death (data not shown). These findings confirm the crucial role of SphK/S1P axis in the induction of differentiation towards SM of these mesenchymal progenitors. Discussion The interplay between extracellular signals and transcriptional regulation is a crucial nexus of control for cell lineage determination. Thus, competence to respond to the milieu of local growth factors is a fundamental determinant of the eventual fate of multipotent cells. In this regard, S1P, that can be locally produced in autocrine manner in response to the cytokine TGFb and many diverse growth factors, deserves considerable attention in view of its pleiotropic mode of action. Remarkably, in this study the analysis of transcriptional profile of human mesoangioblasts in response to S1P treatment not only confirms that this sphingolipid is a powerful anti-apoptotic agent but highlights for the first time its role as morphogenetic cue that, up-regulating GATA6 and LMCD1 transcription factors as well as SM contractile proteins including TPM1, CNN1, TAGLN and CALD1, triggers differentiation of these progenitor cells towards SM phenotype. In addition to the cDNA microarray study, multiple experimental evidence for the pro-differentiating activity of S1P in mesoangioblasts is here provided: the enhanced expression of transcriptional regulators and structural SM-specific proteins was validated by Real time PCR; moreover, Western blotting and confocal microscopy analysis further confirmed the S1P-mediated biological effect. Furthermore, morphological analysis of cells grown on Matrigel revealed that S1P provoked profound phenotypic modifications which were consistent with the accomplishment of a functional SM phenotype. Interestingly, here by siRNA-mediated gene silencing, GATA6 and LMCD1 have been identified as novel transcriptional regulators of mesoangioblast differentiation towards SM. Notably, experimental evidence is provided that this novel molecular mechanism of transcriptional control of SM phenotype appear- ance is integral to the biological action exerted by TGFb in these cells. Modulation of S1P metabolism as well as S1P availability at the site of vascular lesion appears to be highly relevant for switching between different SM phenotypes in physiological and patholog- ical conditions. Indeed, previous studies by Lockman et al. [37] have shown that treating SM cells with S1P increased their proliferation and paradoxically increased the expression of PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20389