Tumor necrosis factor-alpha exerts pro-myogenic action in C2C12 myoblasts via sphingosine kinase/S1P2 signaling.

In this study, we report that low doses of tumor necrosis factor-alpha (TNFalpha) promote myogenesis in C2C12 myoblasts. Moreover, the cytokine increased sphingosine kinase (SphK) activity and induced SphK1 translocation to membranes. The inhibition of SphK functionality by various approaches abrogated the pro-myogenic effect of TNFalpha.

Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts.

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the formation of sphingosine 1-phosphate (S1P), an important lipid mediator, which regulates fundamental biological processes. Here, we provide evidence that SphK is required for the achievement of cell growth arrest as well as myogenic differentiation of C2C12 myoblasts. Indeed, SphK activity, SphK1 protein content and S1P formation were found to be enhanced in myoblasts that became confluent as well as in differentiating cells.

Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor.

In this study a novel biological activity of sphingosine 1-phosphate (S1P) in C2C12 myoblasts was identified. In these cells the bioactive lipid profoundly regulated myogenesis exerting an antimitogenic activity, by reducing serum-induced cell proliferation, and acting as powerful prodifferentiating agent by enhancing the expression of myogenic differentiation markers such as myogenin, myosin heavy chain, and caveolin-3. The S1P-dependent diminution of serum-induced labeled thymidine incorporation was abrogated by antisense oligodeoxyribonucleotides (ODN) to S1P2, but not to S1P1 or S1P3 receptor, also expressed in C2C12 cells, implicating S1P2 in the biological response.

Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D.

The bioactive lipid sphingosine 1-phosphate (S1P) is known to exert powerful biological effects through the interaction with various members of the endothelial differentiation gene (EDG) receptor family, recently renamed S1P receptors. In the present study, evidence is provided that differentiation of C2C12 myoblasts into myotubes was accompanied by profound changes of EDG/S1P receptor expression. Indeed, in differentiated cells a significant increase of EDG3/S1P3 together with a large decrease of EDG5/S1P2 expression at mRNA as well as protein level was detected.

Neutral ceramidase secreted by endothelial cells is released in part associated with caveolin-1.

Neutral ceramidase (CDase) is a key enzyme of sphingomyelin (SM) metabolism implicated in cell signaling triggered by a variety of extracellular ligands. Previously it was shown that in murine endothelial cells a portion of neutral CDase is localized in detergent-resistant light membranes. In this study subcellular distribution of neutral CDase was further investigated.

Sphingosine 1-phosphate signal transduction in muscle cells.

Sphingosine 1-phosphate (S1P) is a powerful bioactive sphingolipid recently recognized to act as extracellular ligand for various subtypes of G protein-coupled receptors belonging to the S1P family. In our study, focused on mouse skeletal muscle cells, we showed that S1P activated enzymes crucial for membrane signal transduction, such as phospholipase D (PLD) and protein kinase C; it promoted also a significant increase of cytosolic Ca2+ via ligation to S1P2 and S1P3 receptor subtypes. Interestingly, myogenic differentiation was found to be accompanied by a profound variation of S1P receptor expression levels and the progressive uncoupling of S1P from PLD activation, suggesting that this signaling pathway is exclusively required for S1P action on proliferating cells.

A role for calcium in sphingosine 1-phosphate-induced phospholipase D activity in C2C12 myoblasts.

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation.