S1P Differentiates Mesoangioblasts Figure 2. Effect of S1P on mRNA (A) and protein (B,C) expression levels of SM markers in human mesoangioblasts. Role of S1PR (D). Cells were incubated in DMEM with S1P and analyzed by A) Real-Time PCR; B) Western blot analysis; (lower panel represents densitometric quantification) and C) Confocal microscopy analysis (406 magnification) using TRITC-phalloidin and anti-fluorescein conjugated specific antibodies. D) Cells were pre-incubated with VPC23017 and JTE071 in DMEM before being challenged with S1P and analyzed by Western blot analysis for CNN1 (lower panel represents densitometric quantification). doi:10.1371/journal.pone.0020389.g002 SKI-2 (Fig. 4C). In order to identify which of the two SphK isoforms is required for the TGFb-induced effect, SphK1 and SphK2 were specifically down-regulated by RNA interference in human mesoangioblasts before being challenged for 48 h with 0.11 mmol/L TGFb. Down-regulation of SphK1 or SphK2 resulted in a significant reduction of the expression of the SM marker CNN1 increased by the cytokine (Fig. 4D), suggesting the involvement of both isoforms in the observed effect. Finally, in order to clarify whether the activation of SphK/S1P axis by TGFb results in the release of the sphingolipid into the medium, we pre-treated mesoangioblasts with 15 mmol/L MK517, an inhibitor of ABCC1 transporter [15], 45 min before challenge with the cytokine. The blockade of ABCC1 transporter completely inhibited the increase of the SM marker TPM1 induced by the cytokine, identifying the transporter as responsible for this autocrine/paracrine pathway in these cells (Fig. 4E). Overall, these latter results support the view that TGFb-induced differentiation of mesoangioblasts towards SM depends on SphK/ S1P axis. In view of the here reported involvement of GATA6 and LMCD1 in the differentiating action of S1P, we next determined whether they were also involved in the mechanism by which TGFb drives differentiation towards SM. As illustrated in Fig. 5A, GATA6 knock-down by specific RNA silencing strongly reduced the TGFb-dependent enhancement of TPM1 mRNA and completely blocked that of the other key myogenic marker CNN1. Similarly, the down-regulation of LMCD1 mRNA abolished the TGFb-induced increase of mRNA encoding for both SM markers TPM1 and CNN1. Interestingly, data presented in Fig. 5B show that TGFb, similarly to S1P, was responsible for an increase of GATA6 mRNA content which was strongly reduced when SphK was pharmacologically inhibited. Likewise, SphK was involved in the increase of LMCD1 mRNA elicited by TGFb (Fig. 5C). Thus, these results highlight a role for GATA6 and LMCD1 in the mechanism by which TGFb exerts its pro- myogenic action. Finally, we sought to evaluate the effect of S1P on morpholog- ical differentiation of mesoangioblasts. To this end, to better explore the phenotypic changes promoted by S1P in human mesoangioblasts, cells seeded in Matrigel and treated with 1 mmol/L S1P were observed at 24 h (Fig. 6A). The addition of the bioactive sphingolipid induced more profound morphological changes compared to unchallenged cells, demonstrating that, in addition to modulate the expression of SM markers, S1P acts as a real differentiating cue in these cells. Since a certain level of differentiation was also appreciable in unchallenged mesoangio- blasts at 24 h, we sought to evaluate whether an endogenously produced S1P by activated SphK might be responsible for the PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20389