Stimulation of protein and DNA synthesis in mouse C2C12 satellite cells: Evidence for phospholipase d‐dependent and ‐independent pathways

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Abstract
In C2C12 myoblasts, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated a phospholipase D (PLD) to degrade phosphatidylcholine (PC) as measured by the release of choline and an increase in the formation of phosphatidic acid (PA) (or phosphatidylbutanol [PtdBuOH] in the presence of 0.5% butanol). Exogenous PLD also stimulated choline release, PA and PtdBuOH formation. The protein kinase C (PKC) inhibitor, Ro-31-8220, and PKC downregulation significantly inhibited the effects of TPA but Ro-31-8220 had no effect on PLD action. Neither basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF) increased PLD activity. All agonists stimulated protein synthesis during both a 90 min and a 6 hr incubation and increased RNA accretion after 6 hr. The response at 90 min was not inhibited by the transcription inhibitor, actinomycin D. Ro-31-8220 and PKC downregulation significantly inhibited all the effects of TPA. In contrast, Ro-31-8220 significantly inhibited the increase in RNA accretion elicited by PLD but had no effect on the ability of agonists other than TPA to enhance protein synthesis. All agonists also stimulated thymidine incorporation into DNA. The effects of EGF, bFGF, and PLD were rapid and transient whereas that of TPA was delayed and sustained. Ro-31-8220 and PKC downregulation significantly inhibited the response due to TPA. Furthermore, Ro-31-8220 also significantly inhibited the effects elicited by EGF and PLD but not that induced by bFGF. In differentiated myotubes, TPA and PLD, but not bFGF or EGF, again stimulated choline release and PtdBuOH formation. However, all agents failed to stimulate protein synthesis and RNA accretion. The data demonstrate the presence in C2C12 myoblasts, but not differentiated myotubes, of both a PLD-dependent and PLD-independent pathway(s) leading to the stimulation of protein synthesis, RNA accretion, and DNA synthesis.