Myoblast undergoes a series of events of proliferation, cell cycle exit, alignment and elongation, fusion and formation of straight muscle fiber during differentiation. Phosphatidylinositol 3-kinase (PI3-kinase) is activated by a variety of extracellular stimuli, which impacts a number of cellular process including cell growth, proliferation, differentiation, migration, and survival. This paper discusses the function of PI3-kinase in proliferation and differentiation of myoblast.
LY294002, an inhibitor of PI3-kinase, decreased the proliferation of L6 myoblasts and delayed the cell cycle progression from G1 to S phase. The expression of cyclin D1 and cdk4 were decreased by LY294002. These results suggest that PI3-kinase regulate the proliferation of L6 myoblasts by promoting the expression of cyclin D1 and cdk4.
When cultured in low growth factor-containing medium, L6 myoblasts exit cell cycle and undergo a well-defined program of differentiation. Phosphorylation of eukaryotic elongation factor 2 (eEF2) was related to the differentiation of chick embryonic muscle cells in culture. The extent of eEF2 phosphorylation declined shortly after differentiation induction of L6 myoblasts, when cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation, however, was blocked by LY294002 and wortmannin, inhibitors of PI3-kinase. These inhibitors have previously been shown to strongly block the differentiation of myoblasts. Therefore, I hypothesized that PI3-kinase plays an important role in the withdrawal from the cell cycle by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, L6 myoblasts were synchronized at the G2/M phase of the cell cycle using nocodazole and then cultured in either fresh differentiation medium (DM) or growth medium (GM). Released cells accumulated in the G0/G1 phase in DM and progressed to the S phase in GM. Cyclin D1 was more rapidly degraded in cells cultured in DM than in GM. The extent of eEF2 phosphorylation was seen to decrease more prominently in DM than in GM. Inhibitors of PI3-kinase or Akt, or mTOR increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.
PI3-kinase induces transcription of myogenin mRNA in myoblast differentiation. Here, I examined another mechanism regulating expression of myogenin by PI3-kinase. When LY294002 was added even after expression of myogenin, the fusion of myoblasts did not increase more than the level at the time treated with LY294002 and myogenin protein was decreased without change of its mRNA level. However, the mRNA and the protein of both GAPDH and b-actin were not changed by LY294002. LY294002 inhibited the rate of global protein synthesis through down-regulation of the activity of S6-kinase, S6, eIF4E, and eEF2. Cycloheximide, inhibitor of translation, rapidly decreased protein level of myogenin without the change of mRNA level but did not affected the level of protein and mRNA of both GAPDH and b-actin. These results shows that a half-life of myogenin protein is greatly shorter than those of GAPDH and b-actin proteins and the inhibition of protein synthesis at the translational level by LY294002 causes rapid depletion of myogenin protein which has a short half-life. This finding represents the first identification that PI3-kinase regulates expression of myogenin post-transcriptionally.
Genistein (4',5,7-trihydroxyisoflavone) is a tyrosine kinase inhibitor. Although the agent has shown to inhibit myoblast differentiation, neither intracellular target(s) as a tyrosine kinase inhibitor nor action mechanism of the agent is well known. Here I studied the effect of genistein on the differentiation of myoblasts. Genistein strongly but reversibly blocked both myoblast fusion and synthesis of the muscle-specific proteins. The agent also reversibly reduced the phosphorylation level of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and its interaction with p85, the regulatory subunit of PI3-kinase. In addition, genistein indirectly inhibited PI3-kinase activity and blocked calcium influx which is required for myoblast fusion. However, both genistein-induced inhibition of cell fusion and calcium influx were abrogated by the lipid products of PI3-kinase. These results demonstrate that genistein can exert their effect on the signaling pathway from FAK to calcium influx via PI3-kinase in the differentiation of myoblasts.
These data, together with the observation that PI3-kinase signaling is involved in the cell cycle progression of myoblast from G1 to S, induction of myogenin, sustaining the expression of myogenin, and cytosolic calcium elevation, suggest that PI3-kinase signaling plays important key roles in the proliferation and the differentiation of myoblasts.