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 cultivat ed in either growth medium or differentiation medium (DM), and the activati on of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parame ters was detected already at 24 h in DM, whereas in cells grown in growth m edium, the increase was evident only after 96 h, when spontaneous different iation was observed in highly crowded cultures. Consequently, we establishe d the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encodi ng human PKR fused to the inducible metallothionein promoter. One of the cl ones (clone 8) expressing high levels of PKR was selected and further analy zed. In the presence of ZnCl2, which activates the promoter, the rate of ce ll growth of the transfected cells was clearly reduced compared to that of wildtype C2C12 cells transfected with only the neomycin-resistant gene (CS- NEO), In addition, altered morphology with partial fusion was observed. Bio chemically, an increase in creatine kinase activity accompanied by an incre ased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells expo sed to ZnCl2, Most importantly, an induction in the level of cyclin-depende nt kinase inhibitor p21(WAF1) and an increase in the level of the underphos phorylated active form of the tumor suppressor protein pRb concomitant with the downregulation of cyclin D1 and c-myc were also evident in the transfe cted clones. These changes were similar to those observed in normal C2C12 c ells cultivated in DM. We conclude that PKR is an important regulatory prot ein participating in the myogenic process.