Inhibition of de novo ceramide synthesis upregulates phospholipase D and enhances myogenic differentiation

Abstract

In L6 skeletal myoblasts induced to differentiate by Arg8-vasopressin treatment, a short-lived lowering of ceramide levels was observed, followed by a long-lasting elevation that was prevented by inhibitors of the de novo synthesis pathway, fumonisin B1 and myriocin. Both inhibitors increased the expression of myogenic differentiation markers and cell fusion rate, whereas short-chain ceramides inhibited these responses. Similar drug effects were observed on primary mouse satellite cell differentiation. Furthermore, bacterial sphingomyelinase overexpression suppressed myogenin nuclear accumulation in L6 cells. These data suggested that endogenous ceramide mediates a negative feedback mechanism limiting myogenic differentiation, and that inhibitors of ceramide synthesis promoted myogenesis by removing this control. Phospholipase D (PLD), a recognized target of ceramide, is required for myogenesis, as shown by the negative effects of PLD1 isoform depletion obtained by siRNA treatment. Fumonisin induced an increase in PLD activity of L6 cells, whereas C6-ceramide decreased it. The expression of PLD1 mRNA transcripts was selectively decreased by C6-ceramide, and increased by ceramide synthesis inhibitors. An early step of myogenic response is the PLD1-dependent formation of actin stress fiber-like structures. C6-ceramide addition or overexpression of sphingomyelinase impaired actin fiber formation. Ceramide might thus regulate myogenesis through downregulation of PLD1 expression and activity.

PLD regulates myoblast differentiation through the mTOR-IGF2 pathway

“…It has been reported that PLD1 is involved in the AVPstimulated myogenic differentiation of L6 rat skeletal myoblasts (Komati et al, 2005;Naro et al, 1997), and that ceramide is a negative regulator in this process upstream of PLD1 (Mebarek et al, 2007). A potentially significant difference between our study and the previously reported ones is that the C2C12 satellite cells undergo differentiation upon growth factor deprivation, in the absence of any hormonal stimulation, which mirrors the myogenic process in normal muscle development and regeneration.…”

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

“…Taking into consideration the emerging evidence of the roles played by SACs (9,43) and cytoskeletal remodeling in skeletal muscle differentiation (9,29), this mechanism of SAC activation may have profound implications in muscle development, regeneration, and diseases, underlying the need to understand the functions of the subplasmalemmal acto-myosin network and its ability to transmit tensional forces to the cell membrane.…”

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

“…The membrane-bound enzyme neutral sphingomyelinase 2 (nSMase2), encoded by the sphingomyelin phosphodiesterase 3 (Smpd3) gene, cleaves sphingomyelin to generate the lipid second messenger ceramide, which affects a variety of cellular process, including proliferation, apoptosis, and differentiation (31)(32)(33)(34). A deletion mutation in the Smpd3 gene was identified in fragilitas ossium (fro), a mouse model of osteogenesis imperfecta (35).…”