title: 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.
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.
authors: Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.
authors: Atossa
authors: A
authors: Shaltouki
authors: Margot
authors: M
authors: Freer
authors: Yu
authors: Y
authors: Mei
authors: Crystal M
authors: CM
authors: Weyman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D052160:Benzothiazoles / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D003470:Culture Media; D045304:Cytochromes c / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D014176:Protein Biosynthesis / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D052160:Benzothiazoles; D003470:Culture Media; C488588:PUMA protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D014050:Toluene; D045304:Cytochromes c; C121565:pifithrin; D053148:Caspase 3; D053453:Caspase 9