Salzberg_1995_Exp.Cell.Res_219_223

Interferon-induced proteins have been previously implicated in the regulation of cell growth. In an attempt to provide evidence for the involvement of these proteins in differentiation, the effect of transforming growth factor beta 1 (TGF-beta) and EGTA on the expression and activity of (2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA activated protein kinase (PKR) during myogenesis of rat primary skeletal muscle cultures or the myogenic cell line L8 was studied. Both TGF-beta and EGTA inhibited the fusion of myoblasts and reduced significantly the level of the muscle-specific proteins, acetylcholine receptors, and creatine kinase activity in rat primary muscle cultures. Likewise, TGF-beta exhibited a similar inhibitory effect on the fusion of L8 cells and the level of creatine kinase activity in these cells. The kinetics of 2-5A synthetase activity in both types of cells during differentiation was then established. In both types, a transient increase in activity was observed followed by a decrease thereafter. However, while the peak activity in primary muscle cultures appeared after 24 h in culture, it was observed only on the third day in L8 cells grown in differentiation medium (DM). Treatment of primary cultures with either TGF-beta or EGTA reduced the amount of 1.7-kb 2-5A synthetase-specific RNA transcripts and decreased significantly the level of 2-5A synthetase activity compared to that in untreated cultures. Western blot analysis of 2-5A synthetase proteins in untreated primary muscle cultures showed that the major species synthesized in these cells was the 43-kDa isoform of the enzyme. However, the 71-kDa isoform was clearly visible after 72 h in culture. Both TGF-beta and EGTA abrogated the appearance of all forms of 2-5A synthetase. Similarly, in L8 cells grown in DM, TGF-beta down-regulated the expression of 2-5A synthetase and reduced the level of enzymatic activity. Western blot analysis revealed the presence of the 71-kDa isoform as the major species of 2-5A synthetase in L8 cells; however, the 43-kDa isoform was also visible on the third day in DM. TGF-beta treatment resulted in a reduced amount of 2-5A synthetase proteins. The kinetics of PKR activity in L8 cells grown in DM was similar to that observed with 2-5A synthetase. Furthermore, TGF-beta strongly reduced the level of PKR activity in differentiating L8 cells.