STIMULATION OF PROTEIN AND DNA-SYNTHESIS IN MOUSE C2C12 SATELLITE CELLS - EVIDENCE FOR PHOSPHOLIPASE-D-DEPENDENT AND PHOSPHOLIPASE-D-INDEPENDENT PATHWAYS

In C2C12 myoblasts, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimula ted a phospholipase D (PLD) to degrade phosphatidylcholine (PC) as mea sured by the release of choline and an increase in the formation of ph osphatidic acid (PA) (or phosphatidylbutanol [PtdBuOH] in the presence of 0.5% butanol). Exogenous PLD also stimulated choline release, PA a nd PtdBuOH formation. The protein kinase C (PKC) inhibitor, Ro-31-8220 , and PKC downregulation significantly inhibited the effects of TPA bu t Re-31-8220 had no effect on PLD action. Neither basic Fibroblast Gro wth Factor (bFGF) or Epidermal Growth Factor (EGF) increased PLD activ ity. All agonists stimulated protein synthesis during both a 90 min an d a 6 hr incubation and increased RNA accretion after 6 hr. The respon se at 90 min was not inhibited by the transcription inhibitor, actinom ycin D. Ro31-8220 and PKC downregulation significantly inhibited all t he effects of TPA. In contrast, Re-31-8220 significantly inhibited the increase in RNA accretion elicited by PLD but had no effect on the ab ility of agonists other than TPA to enhance protein synthesis. All ago nists also stimulated thymidine incorporation into DNA. The effects of EGF, bFGF, and PLD were rapid and transient whereas that of TPA was d elayed and sustained. Re-31-8220 and PKC downregulation significantly inhibited the response due to TPA. Furthermore, Re-31-8220 also signif icantly inhibited the effects elicited by ECF and PLD but not that ind uced 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. T he data demonstrate the presence in C2C12 myoblasts, but not different iated myotubes, of both a PLD-dependent and PLD-independent pathway(s) leading to the stimulation of protein synthesis, RNA accretion, and D NA synthesis. (C) 1995 Wiley-Liss, Inc.