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

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth facto rs and the recruitment of free ribosomes to polysomes was quantified, All e xperiments were conducted on C2C12 myoblasts (24 h prior to induction of fu sion) and differentiating myoblasts (24 h after induction of fusion), After the 2 h incubation, cells were rinsed with phosphate buffered saline and q uickly frozen at - 80 degrees C, Cell lysates were fractionated on 15-60% s ucrose gradients by centrifugation at 200 000 x g for 1 h, Absorbance at 25 4 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 -I, and platelet-derived growth factor was a decrease (P < 0.05) in monosom e 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 respo nse 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 incubati on. Protein content did not vary due to growth factor or level of treatment . This corroborates the hypothesis that the acute increase of protein synth esis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesi s. These results suggest that we can study the mechanisms regulating protei n synthesis in muscle cells effectively by studying shifts in ribosomal act ivity. This method gave more consistent results than the H-3-tyrosine incor poration and has the added benefit of not requiring the use of radioactivit y, The strong correlation between monosome peak heights and percentage poly somes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysom es for other uses. The similarity of response among the various growth fact ors may indicate a common mechanism for increasing the initiation of protei n synthesis, (C) 1999 Harcourt Publishers Ltd.