S1P Differentiates Mesoangioblasts Figure 5. Role of GATA6 and LMCD1 transcription factors on the expression levels of SM markers induced by TGFb in human mesoangioblasts (A) and their dependence on SphK activity (B,C). Cells transfected with scrambled-, GATA6-, LMCD1-siRNA A) and pre- treated with SKI-2 for 45 min (B,C) were stimulated with TGFb and analyzed by Real-Time PCR. doi:10.1371/journal.pone.0020389.g005 of SM-specific proteins identifies this transcription factor as main responsible for differentiation of mesoangioblasts towards SM cells. Intriguingly, GATA6 was recently found involved in the differentiation of mesoangioblasts isolated from ventricular vessel towards cardiomyocytes [45]; interestingly, here, from cDNA microarray study troponin C mRNA was not affected (Supple- mental table S1,S2), nor its protein content examined by Western blot analysis was changed (data not shown). Additionally, in the present study in agreement with a key role of GATA6, TRPS1 which is a potent repressor of GATA transcriptional activity [29] was found dramatically down- regulated by S1P treatment. Moreover, besides GATA6, S1P significantly enhanced mRNA content of a co-regulator of GATA family protein, LMCD1, in accordance with its tissue expression pattern similar to that of GATA6 [30]. Interestingly, here GATA6 and LMCD1 were found both required for the expression of SM- specific proteins and subjected to mutual regulation of their mRNAs. Since LMCD1 has been reported to act also as a co- repressor of GATA6 [30], the present data exclude that this possibility holds true in the mechanism by which S1P promotes SM differentiation of mesoangioblasts, rather supporting its positive role on GATA6 expression. Previously it has been shown that mesoangioblasts express many members of the TGFb/BMP pathway and promptly differentiate into SM cells in response to TGFb [36]. Although serum response cofactor myocardin, MRTFA and Mrf2-a and -b are known to play a fundamental role in SM differentiation [2], TGFb-mediated differentiation of these cells into SM is myocardin-independent [3]. Two transcription factors, Necdin and Msx2, are up-regulated in response to TGFb and are sufficient to induce some SM markers such as SM-specific myosin [3]. From the analysis of the present transcriptional profile, Necdin and Msx2 appear to be not influenced by S1P treatment, which instead was discovered to rely on GATA6 up-regulation for transmitting its biological effect, thus supporting the notion that the complex process of mesoangioblast differentiation into SM is orchestrated by multiple transcription factors. In light of the functional cross-talk that occurs between TGFb and S1P signaling pathways in various cell types [32–35], mesoangioblasts included [12], here the mechanism by which TGFb drives mesoangioblasts towards SM phenotype was further explored. Similarly to what observed for S1P, TGFb was found not only to enhance the expression of GATA6 and LMCD1, but also to depend on these transcriptional regulators for the up- regulation of the SM-specific proteins, TPM1 and CNN1. Importantly, the effect of TGFb on GATA6 and LMCD1 was severely impaired when the biosynthesis of S1P was pharmaco- logically and genetically prevented. Hence, the here identified S1P-dependent novel molecular mechanism for triggering differ- entiation of mesoangioblasts towards SM cells has physiological PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20389