Inositide-dependent phospholipase C signaling mimics insulin in skeletal muscle differentiation by affecting specific regions of the cyclin D3 promoter

Our main goal in this study was to investigate the role of phospholipase C ( PLC) beta 1 and PLC gamma 1 in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLC gamma 1 in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLC beta(1) or PLC beta(1) cDNA and with small interfering RNAs from regions of the PLC beta(1) or PLC gamma(1) gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLC beta(1) and PLC beta(1) were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLC beta(1) or PLC gamma(1) expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5'-deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLC beta(1) or PLC beta(1) cDNAs and small interfering RNAs, respectively. Our data indicate that PLC beta(1) or PLC beta(1) signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLC beta(1) and PLC gamma(1) during myogenic differentiation in vitro and implies that up- regulation of these enzymes is sufficient to mimic the actions of insulin in this process.