A role for PKCepsilon during C2C12 myogenic differentiation.

In a previous report we have demonstrated that PLCgamma1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCgamma1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCepsilon and eta expression, and that PKCepsilon is able to form a complex with PLCgamma1. The increase in PKCepsilon amount during myogenic differentiation is associated to an increase in PKCepsilon activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCgamma1 and PKCepsilon localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCgamma1 and PKCepsilon increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCgamma1-PKCepsilon complex co-localizes with protein 58K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCepsilon form, i.e. PKCepsilon phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCgamma1, we analyzed the effect on cyclin D3 expression of either PKCepsilon overexpression or silencing, in order to investigate whether PKCepsilon could also affect cyclin D3 expression. The results showed that either a modification of PKCepsilon expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCepsilon in regulating insulin inositide-dependent PLCgamma1 signalling in skeletal muscle differentiation.