Up-regulation of nuclear PLCβ1 in myogenic differentiation

Phospholipase Cβ₁ (PLCβ₁) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCβ₁, i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCβ₁ a and PLCβ₁ b Indeed, treatment with insulin induces a dramatic rise of both PLCβ₁ subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti-PLCβ₁ specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCβ₁ expression versus Troponin T expression clearly indicates that the increase in the amount of PLCβ₁ takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCβ₁ M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCβ₁ is a key player in myoblast differentiation, functioning as a positive regulator of this process.