Sphingosine 1-Phosphate Induces Myoblast Differentiation through Cx43 Protein Expression: A Role for a Gap Junction-dependent and -independent Function

Abstract

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43⌬130 -136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

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

“…Cx43 protein is the predominant connexin in skeletal muscle and its involvement in myogenesis and muscle regeneration have been well documented [6,[23][24][25]. We have demonstrated that expression levels of Cx43 are greatly enhanced by S1P in C2C12 skeletal myoblasts and provided the basis for considering the gap junctional protein as an intracellular target of the pro-myogenic action of the sphingolipid [6].…”

“…We have demonstrated that expression levels of Cx43 are greatly enhanced by S1P in C2C12 skeletal myoblasts and provided the basis for considering the gap junctional protein as an intracellular target of the pro-myogenic action of the sphingolipid [6]. Of interest, Cx43 may also perform a gap junction independent function, acting as an adaptor protein through its interaction with F-actin and associated proteins, and establishing an organization center for intracellular signal transduction [6,26,27]. Like many other membrane proteins, Cx43 also exhibits a rapid turnover (half-life of few hours) controlled by a complex interplay of events involving the protein synthesis/degradation, plasma membrane association, and gap junction internalization.…”

“…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?…”

“…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].…”

“…Cx43 protein is the predominant connexin in skeletal muscle and its involvement in myogenesis and muscle regeneration have been well documented [6,[23][24][25]. We have demonstrated that expression levels of Cx43 are greatly enhanced by S1P in C2C12 skeletal myoblasts and provided the basis for considering the gap junctional protein as an intracellular target of the pro-myogenic action of the sphingolipid [6]. Of interest, Cx43 may also perform a gap junction independent function, acting as an adaptor protein through its interaction with F-actin and associated proteins, and establishing an organization center for intracellular signal transduction [6,26,27].…”