S1P Differentiates Mesoangioblasts Figure 1. cDNA microarray analysis. The heat map shows the top 25 down-regulated (left, green) and top 25 up-regulated (right, red) genes. On the left and right side d-values and gene names are reported respectively. Columns 1–2 represent S1P stimulation for 6 h and columns 3–4 represent S1P stimulation for 24 h. doi:10.1371/journal.pone.0020389.g001 was ineffective at a higher concentration, in keeping with the affinity towards S1P exhibited by its receptors. Moreover, we next investigated by Real Time PCR whether the up-regulation of the transcription factor GATA6 and LMCD1 mRNA content elicited by S1P was involved in the induction of the myogenic marker CNN1. As illustrated in Fig. 3B, GATA6 knock-down by specific RNA silencing almost abolished the S1P- dependent enhancement of CNN1 mRNA. The positive effect of S1P on the myogenic marker mRNA levels was also abolished by LMCD1 silencing. In perfect agreement, the down-regulation of GATA6 or LMCD1 mRNA abolished the S1P-induced increase of mRNA encoding for another key myogenic marker such as TPM1 (Fig. 3C). To gain insight into the mechanism by which GATA6 and LMCD1 are necessary for the induction of expression of proteins characteristic of SM phenotype such as CNN1 and TPM1, their mRNA content in response to S1P administration was evaluated when one of the two transcriptional regulators was specifically silenced. Data reported in Fig. 3D show that GATA6 mRNA up-regulation induced by S1P was strongly reduced by LMCD1 silencing; likewise, knock-down of GATA6 was responsible for a markedly diminished effect of S1P on LMCD1 mRNA content (Fig. 3E). Thus, reciprocal regulation of GATA6 and LMCD1 mRNA levels accounts for the observed dual control of SM marker proteins by S1P. The role of GATA6 and LMCD1 in the transcriptional regulation triggered by S1P in mesoangioblasts was further investigated by examining their involvement in the down- regulation of the GATA repressor TRPS1. Specific siRNA targeted to GATA6 did not influence the potent inhibitory effect of S1P on TRPS1 mRNA expression, whereas the efficacy of S1P was strongly attenuated when LMCD1 was silenced, clearly demonstrating that LMCD1 acted also independently from GATA6 regulation (Fig. 3F). The current view indicates that a functional cross-talk between TGFb and S1P signaling occurs in some cell types [32–35]. In this regard, we previously showed that TGFb-dependent SphK/S1P axis is critical for the anti-apoptotic effect of the cytokine in mesoangioblasts [12]. Therefore, since TGFb is a strong inducer of mesoangioblast differentiation towards SM [36], we examined whether SphK might be involved also in this TGFb-induced effect. Indeed, incubation of cells with the specific SphK inhibitor SKI-2 (10 mmol/L) 45 min before TGFb challenge, significantly reduced the increase of TPM1 and completely inhibited that of CNN1 at mRNA level (Fig. 4A). Consistently, at protein level, the blockade of SphK activity markedly reduced the increase of CNN1 and TPM1 (Fig. 4B). Analogously, in these experimental conditions, the cytokine-increased expression of other SM markers, such as TAGLN and laminin was strongly impaired and abolished, respectively (Fig. 4B). In agreement, confocal immunofluorescence analysis revealed that the significant increase of TPM1 and TAGLN expression following treatment with 0.11 mmol/L TGFb was completely inhibited by pre-incubation with 10 mmol/L PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20389