S1P Differentiates Mesoangioblasts Figure 6. Role of SphK/S1P axis on morphogenesis of mesoangioblasts seeded on Matrigel. 206103 cells were plated in Matrigel in the presence of S1P (A) with or without SKI-2 (B) and photographed (106 magnification) at time 0 and after 24 h from plating. doi:10.1371/journal.pone.0020389.g006 relevance since it is exploited by TGFb for exerting its morphogenetic action. Moreover, by specific silencing of SphK1 and SphK2, in human mesoangioblasts the pro-differentiating action of the cytokine was found to depend on both isoforms of SphKs, and the subsequent release of S1P into the media through the ABCC1 transporter as here demonstrated by pre-treatment with the inhibitor MK571 and as already reported in other cell types [15]. However, attempts made to measure S1P into the medium were unsuccessful possibly due to the partition of this sphingolipid into plasma membrane microdomains rather than its release in the extracellular environment (data not shown). Intriguingly, CNN1, here identified downstream of GATA6, is not a downstream target of Necdin or Msx2 in mesoangioblasts challenged with TGFb 3, reinforcing the hypothesis that up- regulation of GATA6 by TGFb is necessary to induce its full biological effect in these cells. It will be interesting to examine in a future study the eventual interplay among Necdin, Msx2 and GATA6 in the regulation of SM phenotype by TGFb in these cells and their hierarchical relationship, possibly extending the investigation to Notch signaling, recently reported to be involved in TGFb-dependent SM differentiation of mesenchymal stem cells [46]. Overall this study increases our knowledge of the role of S1P in these progenitors cells as differentiating cue. Intriguingly, S1P and TGFb, here demonstrated to share molecular mechanisms essential to drive mesoangioblast differentiation, were previously identified as anti-apoptotic cues in these cells [11,12]; moreover, SphK1 up-regulation by TGFb was found critical for the protective role of the cytokine [12]. Whether the prevention of apoptotic death is a determining factor for the successful differentiation of these progenitors cells remains however to be established. As regards the possible intervention in regenerative medicine, in the last few years studies examining the role of S1P on vascular function support the notion that the multiple effects elicited by this sphingolipid could represent potential therapeutic targets for angiogenesis, atherosclerosis and vascular injury. Blood vessel replacement is frequently necessary in the treatment of advanced atherosclerosis and vascular trauma. The emerging field of tissue engineering represents a mean to construct functional grafts when autologous tissue is unavailable due to donor site morbidity. Replacement vessels are engineered in vitro by seeding polymeric tubular scaffolds with autologous endothelial and SM cells [47]. The results of the present study suggest that mesoangioblasts, relatively easy to obtain with an extensive in vitro proliferation capacity, could be potentially used as a source of SM cells for cellular therapeutics and tissue engineering. In conclusion, in this study we demonstrate that S1P induces differentiation of mesoangioblasts mesenchymal stem cells towards a SM phenotype. Importantly, GATA6 up-regulation, together with that of LMCD1, was identified as a novel mechanism of transcriptional regulation that drives SM differen- tiation of mesoangioblasts. Moreover, we provide evidence that GATA6 plays also a role in the mechanism by which TGFb trigger SM differentiation of mesoangioblasts and that S1P biosynthesis is required for the accomplishment of the biological action of the cytokine. The present results represent an original contribution to the knowledge of the molecular mechanisms that regulate differentiation of these progenitors cells and disclose SphK/S1P axis as an attractive novel target to stimulate PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20389