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. 2007 Dec;12(12):2143-54. doi: 10.1007/s10495-007-0135-z.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins
  • Apoptosis* / drug effects
  • Benzothiazoles / pharmacology
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism
  • Cell Differentiation* / drug effects
  • Culture Media
  • Cytochromes c / metabolism*
  • DNA Fragmentation / drug effects
  • Enzyme Activation / drug effects
  • Mice
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Myoblasts, Skeletal / cytology*
  • Myoblasts, Skeletal / drug effects
  • Myoblasts, Skeletal / enzymology
  • Protein Biosynthesis / drug effects
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • Toluene / analogs & derivatives
  • Toluene / pharmacology
  • Tumor Suppressor Protein p53 / antagonists & inhibitors
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Apoptosis Regulatory Proteins
  • Benzothiazoles
  • Culture Media
  • PUMA protein, mouse
  • Proto-Oncogene Proteins c-bcl-2
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Toluene
  • Cytochromes c
  • pifithrin
  • Caspase 3
  • Caspase 9