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

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.

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells

“…There were two main reasons for this. We have observed that both the phospholipase D (Morrison et al, 1995) and insulin (Palmer et al, manuscript in preparation) signal transduction pathways controlling protein synthesis become inoperative upon fusion of C2CI2 cells. Thus, such pathways might also be uncoupled from protein degradation in these cells.…”

Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides

“…This was performed essentially as in previous studies (25,40). Briefly, before each experiment, cells were transferred to 3 ml of serum-free DMEM containing [methyl-3 H]choline (1.33 Ci/ml) for 24 h. At the end of this labeling period, the cells were washed and incubated in DMEM for 30 min.…”

Growth and metabolism of fetal and maternal muscles of adolescent sheep on adequate or high feed intakes: possible role of protein kinase C-α in fetal muscle growth

“…Alterations in muscle DNA, RNA and protein content may involve changes in a number of signalling pathways. Evidence from muscle cell cultures suggests that protein kinase C (PKC) plays a role in the cascade of events by which changes in activity of phospholipases affect accretion of RNA and protein (Morrison et al 1995;Thompson et al 1997) and that one or more of a group of three PKC isofoms (a, 6 and/or E ) may mediate changes in ribosomal activity and protein synthesis in muscle cells in vitro (Thompson et al 1997). This may involve changes in the activity of PKC or in the intracellular redistribution of the enzyme, i.e.…”