Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors

Abstract

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

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

“…Several members of the transient receptor potential canonical (TRPC) ion channel family are recognized as promising candidates for SACs in different cell types [14], including sensory neurons [15], vascular smooth muscle [16], endothelial cells [17], and skeletal myoblasts [4]. Of interest, our recent findings support the involvement of TRPC1/SACs channels in skeletal myogenesis, based on the findings that TRPC1, the isoform mainly expressed in C2C12 cells [4], is sharply up-regulated by stimulation with S1P and, its down-regulation by specific siRNA is able to negatively interfere with SAC-mediated currents as well as with myoblast differentiation and fusion elicited by S1P [4,9]. Moreover, evidence points to a role of TRPC1 in store-operated Ca 2?…”

“…S1P actions on skeletal myoblast differentiation are strictly dependent on intracellular Ca 2? mobilization [6,8,9], actin cytoskeletal remodeling, and mechanosensitive stretch-activated cation channel (SAC) opening [10,11]. The formation of stress fibers by S1P has been associated with plasma membrane stretching and, in turn, with SAC activation in C2C12 myoblasts [12,13].…”

“…entry in skeletal muscle and in the regulation of myoblast migration and fusion [18,19]. Interestingly, store-operated channels have been recently demonstrated to be targets of S1P [8,9]. However, despite the identification of the molecular identity and functional roles of TRPC1 in skeletal myoblasts, little is known about how this channel and its interaction with signaling molecules function within the structural context of living cells to produce the coordinated cellular behaviours required for myoblast differentiation.…”

Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation

“…In view of the previous data underscoring the ability of S1P to promote Ca 2+ release from the intracellular stores (Meacci et al, 2002b;Formigli et al, 2002) and to induce inositol-3-phosphate and diacylglycerol generation leading to store-operated Ca 2+ entry (Blom et al, 2005;Mehta at al., 2005) in several cell types, we asked whether treatment with the bioactive lipid could also affect SOC-dependent transmembrane currents in C2C12 cells. As shown in Table 2, the treatment with 50 μM GdCl3 or 100 μM 2-APB, an inositol-3-phosphate receptor-dependent SOC inhibitor (Lievremont et al, 2005), reduced G m /C m by approximately 27% and 40% in unstimulated cells, respectively, and by approximately 60% and 65% in S1P-stimulated cells.…”

“…An important sphingolipid metabolite is sphingosine 1-phosphate (S1P), which mainly acts through Gprotein-coupled receptors present on mammalian cells, thereby regulating numerous cell functions, including cell proliferation, differentiation and apoptosis (Zeidan and Hannun, 2007). In the last few years, most of the studies of our group have been focused on the role played in skeletal muscle cell biology by S1P, as a modulator of intracellular Ca 2+ mobilization, phosphatidic acid synthesis and phospholipid remodelling in skeletal muscle cells (Bencini et al, 2003;Donati et al, 2005;Formigli et al, 2002;Meacci et al, 1999;Meacci et al, 2002a;Meacci et al, 2002b). Recently, we have underscored the physiological relevance of the sphingosine-kinase-S1P axis in C2C12 cell growth arrest and differentiation and identified the essential steps of the pro-myogenic action of S1P in skeletal myoblasts, including formation of SFs, upregulation of connexin 43 protein and its interaction with cortical actin (Formigli et al, 2007b;Squecco et al, 2006).…”