The IGF-1 signaling pathway plays a key role in the regulation of skeletal muscle growth, in differentiation, and in the maintaining of tissue homeostasis. Intriguingly, this growth factor stimulates two key processes such as myoblast proliferation and differentiation which are mutually exclusive events during myogenesis . The results presented here for the first time demonstrate that the SK/S1P signaling pathway is involved in the regulation of the biological action exerted by IGF-1. This finding is crucial for the timely regulation of cell proliferation and differentiation. Indeed, experimental evidence is provided that activation of SK followed by transactivation of S1P2 elicited by the growth factor is required for its pro-myogenic action whereas the parallel engagement of S1P1 and S1P3 reduces its mitogenic effect. Notably, the IGF1-induced SK/S1P axis appears to exhibit the unique property of regulating two opposite biological effects elicited by IGF-1 in myoblasts - transducing its myogenic response on one side and inhibiting its mitogenic effect on the other - therefore acting as an efficacious switch to stop proliferation and triggering differentiation through the concomitant activation of individual S1PR subtypes.
Previously we have demonstrated in these cells that the final biological outcome of PDGF, which plays a crucial role in skeletal muscle, depends on SK/S1P axis activation . Indeed, the activation of SK1 induced by PDGF resulted in the transactivation of S1P1, responsible for the chemotactic response elicited by PDGF and also for the negative regulation of its mitogenic effect. Despite the overlapping role of the SK/S1P axis in inhibiting the mitogenic effect of both PDGF and IGF-1, we show here that the negative regulation of IGF-1-induced proliferation, beside being dependent on S1P1, relies also on S1P3 engagement. Moreover, in this study, we have established that the IGF-1 myogenic effect is dependent on the engagement of S1P2, which has been already shown to be coupled to the pro-differentiating activity of exogenous and endogenous S1P in these cells [7, 22].
Interestingly, here we provide the first evidence of the involvement of SK2 in S1P inside-out signaling in myoblasts. Indeed SK2, similarly to the SK1 isoform, was found to be required for transmitting the pro-myogenic effect of IGF-1, highlighting a key role of this enzyme isoform in skeletal muscle biology. In this regard, the parallel regulation of mitogenesis and migration elicited by PDGF was found to rely exclusively on the specific activation of SK1 . SK1 and SK2 have different developmental expression, adult tissue distribution and subcellular localization patterns. Such differences are in line with the concept that, although the two enzymes use the same substrate and generate the same product, they can mediate distinct biological events. SK1 is almost universally associated with the induction of cell survival and proliferation , whereas SK2 overexpression suppresses cell growth and enhances apoptosis . Studies performed in mesangial cells supported the pro-apoptotic role of SK2, demonstrating that SK2-null cells are more resistant to staurosporine-induced apoptosis than wild-type or SK1-null cells . Our findings corroborate the notion that SK isoforms may instead have overlapping functions. Indeed, data obtained by preincubation with the pharmacological inhibitor SKI-2 that does not distinguish between the two SK isoforms, and by specific silencing of SK1 or SK2 isoforms demonstrate the involvement of both enzyme isoforms in the pro-myogenic effect of IGF-1. In accordance, it has been recently reported that SK1 and SK2 are equally required for epidermal growth factor-induced migration of breast cancer cells  and TGFβ-induced migration and invasion of esophageal cancer cells .
Data previously reported in the literature sustain the existence of a functional cross-talk between IGF-1 and S1P signaling pathways. IGF-1 was shown to elicit ERK1/2 activation by stimulating SK1-dependent transactivation of S1P1 even though the biological effect of S1P-dependent ERK1/2 activation was not addressed [28, 29]. In this study, for the first time, we identified the involvement of the SK/S1PR axis in the biological action of IGF-1, that intriguingly in its molecular mechanism of action implies ERK1/2-dependent phosphorylation of SK1.
It is worth noting that exogenous S1P has been previously reported to counteract the chemotactic action exerted by IGF-1 in myoblasts via ligation of S1P2. The involvement reported in this paper of the SK/S1P axis in mediating IGF-1 biological effects is not in constrast with the previous finding. Indeed, it appears that the selective spatial control of S1P formation inside myoblasts following stimulation with different cues is crucial for the generation of a specific biochemical response. In this regard, we recently demonstrated that endogenous S1P that is formed in response to PDGF challenge stimulates cell motility via S1P1, whereas exogenous S1P was previously found to inhibit motility of the same cell type acting through S1P2. Moreover, we recently demonstrated that, in C2C12 myoblasts, the S1P signaling pathway, which physiologically accounts for myogenic differentiation via S1P2, is redirected by the cytokine TGFβ to act primarily via the pro-fibrotic S1P3. Indeed, consequently to TGFβ treatment, S1P3 then becomes the prevailing expressed receptor, thus promoting myoblast transdifferentiation to myofibroblasts .
Finally, we investigate the possible involvement of SPL in skeletal muscle biology. A previous study performed by Herr and colleagues  demonstrated that normal S1P catabolism is required for Drosophila muscle development, given that SPL-null mutants exhibited pattern abnormalities in the dorsal longitudinal flight muscles of the adult thorax. Moreover, SPL was recently identified as a potential therapeutic target for ischemia-reperfusion injury of the heart . Using a genetic SPL knock-out mouse model and a chemical inhibitor, Bandhuvula and colleagues  demonstrated that ischemia caused the activation of SPL in cardiac tissue resulting in the reduction of the levels of S1P, thus promoting cardiomyocyte apoptosis. For the first time, we have demonstrated in this paper that mouse myoblasts express SPL. Furthermore, we provide evidence that this enzyme contributes to the regulation of intracellular levels of the pro-myogenic S1P, as knocking-down of the enzyme resulted in the upregulation of the basal expression levels of myogenic markers. This is in agreement with the concept that, in this experimental condition, S1P intracellular levels are enhanced and consequently the mediated-promyogenic action is potentiated [7, 22]. However, from this study, SPL appears to be disengaged from IGF-1-mediated biological effects as its specific downregulation did not affect either myogenesis or proliferation induced by the growth factor.
Taking into account the different S1PR patterns expressed in satellite cells and murine myoblasts, and considering that exogenous S1P is mitogenic in satellite cells whereas it acts as an anti-mitogenic and pro-differentiating cue in myoblasts [6, 7], it will be interesting to further investigate whether the individuated cross-talk between the IGF-1 and S1P signaling pathway observed here also takes place in satellite cells and mediates a specific IGF-1-induced biological response