Expression of a PKR dominant-negative mutant in myogenic cells interferes with the myogenic process.
In this study, we explored the possibility that PKR, a dsRNA-activated regulatory protein, is an essential component in the differentiation program of myogenic cells in vitro. For this purpose, we used a retroviral expression vector pMV7, harboring the PKR dominant-negative mutant PKRDelta6 (pMV7-p68Delta6). Murine C2C12 myogenic cells were transfected either with pMV7 or with pMV7-p68Delta6. Neomycin-resistant clones from both types were isolated and expanded and the results obtained with the representative clones C2-NEO (transfected with pMV7) and clone 17 and clone 22 (both transfected with pMV7-p68Delta6) are presented. In clone 17 and 22 cells, regardless of IFN treatment, a similar level of the transfected human p68 PKR mutant was detected. This protein was absent in C2-NEO cells. In parallel, in all types of cells, a low basal level of the endogenous murine p65 PKR protein was observed, which was further induced by IFN. However, PKR enzymatic activity was significantly induced by IFN only in C2-NEO cells, while it was hardly detected in both clones 17 and 22, even after IFN treatment. Furthermore, in contrast to C2-NEO cells, only a slight to moderate increase in enzymatic activity was observed in clone 17 and 22 differentiating cells. Next, cells were grown either in growth medium (GM) or differentiation medium (DM), and the progression of the myogenic program was studied. An inhibition in myotube formation in clone 17 versus C2-NEO cells cultivated in DM was clearly observed. Furthermore, while the growth rate and thymidine incorporation were reduced in C2-NEO cells grown in DM, both clone 17 and 22 cells were less affected under the same conditions. Similarly, a delay in the accumulation of the transcription factors MyoD and myogenin, as well as in creatine kinase activity and accumulation of troponin T, was detected in DM-cultivated clone 17 and clone 22 cells. Moreover, a delay in the induction of p21 (WAF1), in down-regulation of cyclin D1 and c-myc, and in the accumulation of the underphosphorylated form of pRb was also observed in clone 17 cells. We conclude that inhibition of endogenous PKR activity by a PKR dominant-negative mutant interferes with the myogenic program of murine C2C12 myogenic cells.
Salzberg
S
S
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. salzbs@mail.biu.ac.il
Vilchik
S
S
Cohen
S
S
Heller
A
A
Kronfeld-Kinar
Y
Y
D000818:Animals; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002999:Clone Cells; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D009132:Muscles / Q000166:cytology*; D016296:Mutagenesis; D017570:MyoD Protein / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017570:MyoD Protein; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019892:eIF-2 Kinase