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 gamma, 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 gamma, a member of the DNase I family of DNases, is correlated with the appearance of inducible DNase activity. The induction of DNase gamma occurs simultaneously with myogenin induction but precedes the up-regulation of p21. A high level of DNase gamma expression was also detected in differentiated myotubes but not in skeletal muscle fibers in which DNase X is highly expressed. The role of DNase gamma in myoblast apoptosis was evaluated in the following experiments. Proliferating myoblasts acquire DNA ladder producing ability by the ectopic expression of DNase gamma, but not DNase X, suggesting that the expression level of DNase gamma is the determinant of the differentiation-dependent apoptotic DNA laddering observed in myoblasts. DNA fragmentation during differentiation-induced apoptosis is strongly suppressed by the antisense-mediated down-regulation of DNase gamma. Importantly, the extent of DNA laddering is well correlated with the level of endogenous DNase gamma activity. Our data demonstrate that DNase gamma is the endonuclease responsible for DNA fragmentation in apoptosis associated with myogenic differentiation.
Highlights
Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions
We have found that DNase ␥ is newly synthesized via transcriptional activation in C2C12 cells during myogenic differentiation and that interference in DNase ␥ induction by the expression of its antisense RNA markedly suppresses apoptotic DNA fragmentation in differentiating myoblasts
Western blot confirmed the same expression levels of DNase ␥ and DNase X in each transfectant (Fig. 5D). These results clearly indicate that the ectopic expression of DNase ␥ enables proliferating C2C12 cells to undergo DNA ladder formation and suggest that the expression of DNase ␥ is the cause of the differentiation-dependent DNA fragmentation observed in C2C12 apoptosis
Summary
Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions. We show that a Ca2؉/Mg2؉-dependent endonuclease, DNase ␥, is induced in C2C12 myoblasts during myogenic differentiation and catalyzes apoptotic DNA fragmentation in differentiating myoblasts. Murine C2C12 myoblasts have been widely used as a model system in which to study apoptosis in developing muscle, because extensive cell death occurs during myogenic differentiation [13, 14]. C3H-10T1/2 cells, which are unable to degrade genomic DNA into nucleosomal fragments, acquire ladder producing ability when myogenic differentiation is induced by the forced expression of MyoD [15]. The elucidation of the mechanism of differentiation-dependent DNA fragmentation is important for understanding apoptosis in developing muscles; the molecular basis for the interesting observations is as yet not well understood
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