Catalytic activity of nuclear PLC-β1 is required for its signalling function during C2C12 differentiation

Here we report that PLC-β₁ catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-β₁ mutational analysis revealed the importance of His³³¹ and His³⁷⁸ for the catalysis. The expression of PLC-β₁ and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-β₁ activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β₁ is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β₁ catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-β₁ that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-β₁ and that c-Jun binding activity is significantly increased by insulin and PLC-β₁ overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-β₁. These results hint at the fact that PLC-β₁ catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.