Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy.

Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle.

Isolation of a differentiation-defective myoblastic cell line, INC-2, expressing muscle LIM protein under differentiation-inducing conditions.

A non-differentiating myoblastic cell line, INC2, and a differentiating cell line, COM3, were established from the mouse myoblastic cell line C2C12. Under differentiation conditions, both COM3 and INC2 cells stopped proliferation in a similar manner. The COM3 cells then differentiated into myotubes during the 4-day differentiation culture.

Role of tyrosine kinase in the regulation of myogenin expression.

Using an affinity-purified anti-myogenin antibody, three stages of mouse myoblast C2C12 cells during myogenesis could be identified: proliferating myoblasts as myogenin-negative mononucleated cells, differentiating myoblasts as myogenin-positive mononucleated cells, and myotubes as myogenin-positive multinucleated cells. We found differential effects of genistein, an inhibitor of protein-tyrosine kinase, on myogenic cells during these three stages. Genistein severely inhibited myotube formation and myogenin production in differentiating myoblasts by inhibiting the transcription of the myogenin gene in a dose-dependent manner.

Phosphorylation of a proline-directed kinase motif is responsible for structural changes in myogenin.

Myogenin, a member of the MyoD family which governs skeletal muscle differentiation, was identified as a pair of phosphorylated bands on SDS-PAGE during myogenesis. The slow migrating form was found to be hyperphosphorylated myogenin. In vitro phosphorylation by CDC2 kinase caused a prominent reduction in electrophoretic mobility of myogenin.