S1P Differentiates Mesoangioblasts Figure 4. Effect of SphK inhibition (A,B,C), dowregulation (D) and ABCC1 transporter inhibition (E) on the expression levels of SM markers induced by TGFb treatment in human mesoangioblasts. Cells were pre-treated with SKI-2 45 min before being incubated with DMEM for 48 h with TGFb and analyzed by A) Real-Time PCR analysis B) Western blot analysis and C) Confocal microscopy analysis (636 magnification) using anti fluorescein-conjugated specific antibodies. D) Cells were transfected with scrambled-, SphK1-, SphK2-siRNA, stimulated with TGFb and analyzed by Western blot analysis for CNN1. Lower panel represents densitometric quantification. E) Cells were pre-treated with MK571 45 min in DMEM before being incubated with TGFb for 48 h and analyzed by Western blot analysis for TPM1. Lower panel represents densitometric quantification. doi:10.1371/journal.pone.0020389.g004 SM-specific a-actin and myosin as well as TAGLN. Subsequently, Wamhoff et al. demonstrated that S1P receptors are differentially affected by vascular injury and that S1P1/3 negatively regulate the phenotypical appearance of contractile protein and positively regulate proliferation, while, S1P2 opposes these actions [38]. Here, on the contrary, by pharmacological inhibition, S1P2 and S1P1/3 were found to be involved in the differentiating action of S1P towards SM of these progenitor mesodermal cells, in agreement with what previously reported in adipose tissue-derived mesenchymal stem cells where S1P2 and, at a lesser extent S1P3, turned out to be critical for the pro-differentiating effect of S1P [20]. The role of S1P2 in SM differentiation was also recently confirmed since its activation was required for the expression of SM-specific a-actin after arterial injury [39] and LARG-mediated RhoA activation leading to SM differentiation [40]. Additionally, the expression of SM-specific a-actin induced by TGFb in C2C12 myoblasts was found to be downstream of S1P/S1P3 axis [41]. Moreover, S1P induces Ca2+-dependent contraction, prolifera- tion and migration of SM cells [9]. Finally, genetically mice for S1P1 are embryonically lethal due to impaired homing of SM cells to developing blood vessels, highlighting the importance of the S1P signalling via S1P1 for vascular SM cell migration [42]. 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. This is not only due to its multiple effects on cardio-vascular system but also because S1P plays an important role in the regulation of embryonic and adult stem cells [43]. In this regard S1P appears to play a dual role, since it maintains human embryonic stem cells in undifferentiated state [10] and acts as a differentiating cue in toti- or multi-potent cells: it induces cardiac differentiation of embryoid bodies derived from mouse embryonic stem cells [44] and up-regulates the expression of SM marker proteins of adipose tissue-derived mesenchymal stem cells [20], reinforcing the notion of a pleiotropic action of this lipid. Despite this conspicuous amount of information, little is known about the eventual transcription regulators that orchestrate the expression of tissue- specific proteins under the control of S1P, with the notable exception of cofactors of the myocardin family found implicated in differentiation of vascular rat SM cells [37]. The present finding that knock-down of GATA6 abrogates the S1P-induced expression PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20389