Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.
Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.
Whiteman
Matthew
M
Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Dr., Republic of Singapore 117597. bchwml@nus.edu.sg
Armstrong
Jeffrey S
JS
Cheung
Nam Sang
NS
Siau
Jia-Ling
JL
Rose
Peter
P
Schantz
Jan-Thorsten
JT
Jones
Dean P
DP
Halliwell
Barry
B
D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000494:pharmacology*
D030421:Peroxynitrous Acid; D002154:Calpain; D002118:Calcium