Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts

Abstract

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at – 80°C. Cell lysates were fractionated on 15–60% sucrose gradients by centrifugation at 200 000 × g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P< 0.05) in monosome peak height and a increase (P< 0.05) in polysome percentage (P< 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P< 0.05). None of the growth factors had a significant effect (P> 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.