Involvement of PKR in the Regulation of Myogenesis

The involvement of the double-stranded RNA-activated protein kinase PKR in the regulation of the myogenic process was investigated. For this purpose, the murine myogenic cell line C2C12 was used. The cells were first cultivated in either growth medium or differentiation medium (DM), and the activation of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parameters was detected already at 24 h in DM, whereas in cells grown in growth medium, the increase was evident only after 96 h, when spontaneous differentiation was observed in highly crowded cultures. Consequently, we established the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encoding human PKR fused to the inducible metallothionein promoter. One of the clones (clone 8) expressing high levels of PKR was selected and further analyzed. In the presence of ZnCl 2, which activates the promoter, the rate of cell growth of the transfected cells was clearly reduced compared to that of wildtype C2C12 cells transfected with only the neomycinresistant gene (C2-NEO). In addition, altered morphology with partial fusion was observed. Biochemically, an increase in creatine kinase activity accompanied by an increased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells exposed to ZnCl 2. Most importantly, an induction in the level of cyclin-dependent kinase inhibitor p21 WAF1 and an increase in the level of the underphosphorylated active form of the tumor suppressor protein pRb concomitant with the downregulation of cyclin D1 and c-myc were also evident in the transfected clones. These changes were similar to