The pleiotropic cytokine transforming growth factor (TGF)-β1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFβ1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFβ1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFβ1. Moreover, inhibition of S1P₃, which became the highest expressed S1PR after TGFβ1 challenge, strongly attenuated the profibrotic response to TGFβ1. Furthermore, downstream of S1P₃, Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFβ1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P₂, consequently to TGFβ1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFβ1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.

Mesoangioblasts are stem cells capable of differentiating in various mesodermal tissues and are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases, as well as myocardial infarction. The enhancement of their proliferation and survival after injection in vivo could greatly improve their ability to repopulate damaged tissues. In this study, we show that the bioactive sphingolipid sphingosine 1-phosphate (S1P) regulates critical functions of mesoangioblast cell biology. S1P evoked a full mitogenic response in mesoangioblasts, measured by labeled thymidine incorporation and cell counting. Moreover, S1P strongly counteracted the apoptotic process triggered by stimuli as diverse as serum deprivation, C2-ceramide treatment, or staurosporine treatment, as assessed by cell counting, as well as histone-associated fragments and caspase-3 activity determinations. S1P acts both as an intracellular messenger and through specific membrane receptors. Real-time polymerase chain reaction analysis revealed that mesoangioblasts express the S1P-specific receptor S1P3 and, to a minor extent, S1P1 and S1P2. By using S1P receptor subtype-specific agonists and antagonists, we found that the proliferative response to S1P was mediated mainly by S1P2. By contrast, the antiapoptotic effect did not implicate S1P receptors. These findings demonstrate an important role of S1P in mesoangioblast proliferation and survival and indicate that targeting modulation of S1P-dependent signaling pathways may be used to improve the efficiency of muscle repair by these cells. Disclosure of potential conflicts of interest is found at the end of this article.

In view of its multiple detrimental effects, transforming growth factor β1 (TGFβ1) is recognized as critical negative regulator of skeletal muscle repair. Apoptosis of skeletal muscle precursor cells driven by TGFβ1 contributes to the negative role exerted by the cytokine in tissue repair, although the underlying molecular mechanisms are still elusive. Herein we report the identification of a new signaling pathway, relying on Rho kinase‐2 stimulation, subsequent to SMAD‐dependent S1P ₄ up‐regulation and transactivation via sphingosine kinase (SK)‐2, that accounts for TGFβ1‐induced apoptosis in cultured myoblasts. S1P ₄ ‐specific gene silencing reduced by almost 50% activation of caspase‐3 and poly‐ADP ribosyl transferase cleavage elicited by TGFβ1. Moreover, the selective S1P ₄ antagonist CYM50358 also reduced the TGFβ1 proapoptotic effects. By employing pharmacological and molecular biological approaches, the involvement of SK2 and ROCK2 in the transmission of the TGFβ1 apoptotic action was also demonstrated. These results reinforce the notion that the SK/S1P axis plays a fundamental role in TGFβ1 mode of action in skeletal muscle cells and, by disclosing a novel mechanism by which TGFβ1 exerts its harmful action, pinpoint new molecular targets that in principle could be beneficial in the treatment of several skeletal muscle disorders or aging‐dependent muscle atrophy.—Cencetti, F., Bernacchioni, C., Tonelli, F., Roberts, E., Donati, C., Bruni, P. TGFβ1 evokes myoblast apoptotic response via a novel signaling pathway involving S1P ₄ transactivation upstream of Rho‐kinase‐2 activation. FASEB J . 27, 4532–4546 (2013). www.fasebj.org