Prostaglandin E(2) promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E(2) (PGE(2)), a bioactive lipid mediator in various physiological or pathological conditions. PGE(2) is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE(2) signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE(2) or specific agonists. All four PGE(2) receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE(2) and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE(2) (17-PT PGE(2)) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE(2) signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.