Native, not nitrated, cytochrome c and mitochondria-derived hydrogen peroxide drive osteoclast apoptosis.
Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H(2)O(2) production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H(2)O(2) production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H(2)O(2). Trapping H(2)O(2)-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts.
Oursler
Merry Jo
MJ
Department of Biology, University of Minnesota, Duluth, Minnesota, USA. Oursler.MerryJo@mayo.edu
Bradley
Elizabeth W
EW
Elfering
Sarah L
SL
Giulivi
Cecilia
C
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D010084:Oxidation-Reduction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D005609:Free Radicals; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D020169:Caspases; D009584:Nitrogen