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Increased expression of the pro-apoptotic Bcl2 family member PUMA is required for mitochondrial release of cytochrome C and the apoptosis associated with skeletal myoblast differentiation

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Abstract

We have previously shown that when skeletal myoblasts are cultured in differentiation medium (DM), roughly 30% undergo caspase 3-dependent apoptosis rather than differentiation. Herein, we investigate the molecular mechanism responsible for the activation of caspase 3 and the ensuing apoptosis. When 23A2 myoblasts are cultured in DM, caspase 9 activity is increased and pharmacological abrogation of caspase 9 activation impairs caspase 3 activation and apoptosis. Further, we detect a time dependent release of mitochondrial cytochrome C into the cytosol in roughly 30% of myoblasts. Inclusion of cycloheximide inhibits the release of cytochrome C, the activation of caspase 9 and apoptosis. These data indicate that the mitochondrial pathway plays a role in this apoptotic process and that engagement of this pathway relies on de novo protein synthesis. Through RT-PCR and immunoblot analysis, we have determined that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated when 23A2 myoblasts are cultured in DM. Further, silencing of PUMA inhibits the release of cytochrome C and apoptosis. Signaling by the transcription factor p53 is not responsible for the increased level of PUMA. Finally, myoblasts rescued from apoptosis by either inhibition of elevated caspase 9 activity or silencing of PUMA are competent for differentiation. These results indicate a critical role for PUMA in the apoptosis associated with skeletal myoblast differentiation and that a p53-independent mechanism is responsible for the increased expression of PUMA in these cells.

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Acknowledgements

We thank Andrei Gudkov (Lerner Research Institute, Cleveland Clinic) for generously providing constructs and assistance with the lenti-viral silencing. This work was supported by NIH grant RO1CA84212 awarded to C. M. Weyman. A. Shaltouki and M. Freer were each supported by a Molecular Medicine Fellowship and by the President’s Initiative Fund provided by Cleveland State University.

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Authors and Affiliations

  1. Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH, 44115, USA

    Atossa Shaltouki, Yu Mei & Crystal M. Weyman

  2. Department of Biological Sciences, Kent State University-Salem, Salem, OH, 44460, USA

    Margot Freer

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  1. Atossa Shaltouki
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  2. Margot Freer
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Correspondence to Crystal M. Weyman.

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Shaltouki, A., Freer, M., Mei, Y. et al. Increased expression of the pro-apoptotic Bcl2 family member PUMA is required for mitochondrial release of cytochrome C and the apoptosis associated with skeletal myoblast differentiation. Apoptosis 12, 2143–2154 (2007). https://doi.org/10.1007/s10495-007-0135-z

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