Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts

Abstract

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. Enforced expression of SphK1 reduced the myoblast proliferation rate, enhanced the expression of myogenic differentiation markers and anticipated the onset of differentiated muscle phenotype. Conversely, down-regulation of SphK1 by specific silencing by RNA interference or overexpression of the catalytically inactive SphK1, significantly increased cell growth and delayed the beginning of myogenesis; noticeably, exogenous addition of S1P rescued the biological processes. Importantly, stimulation of myogenesis in SphK1-overexpressing myoblasts was abrogated by treatment with short interfering RNA specific for S1P(2) receptor. This is the first report of the role of endogenous SphK1 in myoblast growth arrest and stimulation of myogenesis through the formation of S1P that acts as morphogenic factor via the engagement of S1P(2).

Sphingosine-1-phosphate: distribution, metabolism and role in the regulation of cellular functions

“…Migratory capability of myoblasts is a property of great importance necessary for myogenesis. In compliance with data on S1P's significance in myogenesis regu lation, enhanced SphK1 expression in confluent and differentiating C2C12 myoblasts, as well as an arrest of S1P(2)-mediated myoblast growth were revealed [120] (Fig. 4).…”

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

“…A non-specific scrambled (SCR) siRNA was used as the control. C2C12 myoblasts at 80% confluence were transfected using Lipofectamine 2000 reagent (1 mg/ml; Invitrogen) with TRPC1-or calpain-siRNA duplexes or with SCR-siRNA (20 nM) for 24 h, as previously reported [4,5]. The specific knock-down of TRPC1 and m-calpain were evaluated by Western blotting or enzymatic assay or confocal immunofluorescence.…”

“…Its role in skeletal muscle cell biology is beginning to be highlighted by recent data showing that the sphingolipid is able to promote satellite cell activation and proliferation [2,3], regulate myogenic differentiation [4][5][6], and exert a trophic action on denervated muscle [7]. S1P actions on skeletal myoblast differentiation are strictly dependent on intracellular Ca 2?…”

Tumor necrosis factor‐α exerts pro‐myogenic action in C2C12 myoblasts via sphingosine kinase/S1P2 signaling

“…For experiments confluent cells were shifted to DMEM without serum containing 1 mg/ml BSA. Cells stably expressing WTSphK1 or DNSphK1 were obtained as described previously [7,13]. To silence SphK1 and S1P 2 , cells were transfected with specific or scrambled siRNA using Oligofectamineä reagent according to manufacturer's instructions.…”

“…We have previously shown that S1P promotes myogenic differentiation of C2C12 myoblasts [6]. Moreover, sphingosine kinase (SphK), which catalyzes the phosphorylation of sphingosine to S1P, has been recently found to be required for myogenic differentiation [7]. Since various studies have demonstrated that SphK is involved in the signaling pathway of TNFa and in its subsequent biological response [8][9][10], the possible involvement of SphK/ S1P signaling in the pro-myogenic effect exerted by low dose of TNFa has been here investigated.…”