Ruolo della fosfolipasi C-?1 nel differenziamento miogenico: identificazione di nuovi target nucleari

The expression of phospholipase C-?1 (PLC-?1) and cyclin D3 is highly induced during skeletal myoblast differentiation. We have previously shown that PLC-?1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-?1 is a crucial regulator of mouse cyclin D3 gene. Here we report that PLC-?1 catalytic activity plays a role in the increase of cyclin D3 levels and in the induction of differentiation of C2C12 skeletal muscle cells. PLC-?1 mutational analysis revealed the importance of His331 and His378 for the catalytic activity. We show that following insulin administration, cyclin D3 mRNA levels are lower in cells overexpressing the PLC-?1 catalytically inactive form, as compared to wild type cells. We describe a novel signaling pathway elicited by PLC-?1 that modulates Activator Protein-1 (AP-1) activity. Indeed, gel mobility shift assays indicate that there is a c-jun binding site located in cyclin D3 promoter region specifically regulated by PLC-?1 and that c-jun binding activity is significantly increased by insulin stimulation and PLC-?1 overexpression. Moreover, mutation of c-jun/AP-1 binding site decreases the basal cyclin D3 promoter activity and eliminates its induction by insulin and PLC-?1 overexpression. Interestingly, we observed that the ectopic expression of the Inositol Polyphosphate Multikinase (IPMK) in C2C12 myoblasts enhances cyclin D3 gene expression and that the mutation of c-jun site in cyclin D3 promoter determines an impairment of IPMK-dependent promoter induction. These results indicate that PLC-?1 activates a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation through IPMK signaling.