Involvement of DNase γ in apoptosis associated with myogenic differentiation of C2C12 cells

Abstract

Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions. However, the molecular mechanisms and the DNase responsible for the differentiation-dependent apoptotic DNA laddering are poorly understood. Here we show that a Ca 2+/Mg 2+-dependent endonuclease, DNase γ, is induced in C2C12 myoblasts during myogenic differentiation and catalyzes apoptotic DNA fragmentation in differentiating myoblasts. A Ca 2+/Mg 2+-dependent, Zn 2+-sensitive endonuclease activity appears in C2C12 myoblasts during myogenic differentiation. The enzymatic properties of the inducible DNase were found to be quite similar to those of DNase I family of DNases. Reverse transcriptase-PCR analysis revealed that the induction of DNase γ, a member of the DNase I family of DNases, is correlated with the appearance of inducible DNase activity. The induction of DNase γ occurs simultaneously with myogenin induction but precedes the up-regulation of p21. A high level of DNase γ expression was also detected in differentiated myotubes but not in skeletal muscle fibers in which DNase X is highly expressed. The role of DNase γ in myoblast apoptosis was evaluated in the following experiments. Proliferating myoblasts acquire DNA ladder producing ability by the ectopic expression of DNase γ, but not DNase X, suggesting that the expression level of DNase γ is the determinant of the differentiation-dependent apoptotic DNA laddering observed in myoblasts. DNA fragmentation during differentiation-induced apoptosis is strongly suppressed by the antisensemediated down-regulation of DNase γ. Importantly, the extent of DNA laddering is well correlated with the level of endogenous DNase γ activity. Our data demonstrate that DNase γ is the endonuclease responsible for DNA fragmentation in apoptosis associated with myogenic differentiation.