Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization.
OBJECTIVE
Increased chondrocyte nitric oxide (NO) and peroxynitrite production appears to modulate decreased matrix synthesis and increased mineralization in osteoarthritis (OA). Because NO inhibits mitochondrial respiration, this study was undertaken to directly assess the potential role of chondrocyte mitochondrial oxidative phosphorylation (OXPHOS) in matrix synthesis and mineralization.


METHODS
We studied cultured human articular chondrocytes and immortalized costal chondrocytes (TC28 cells). We also assessed the effects of antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) on chondrocyte mitochondrial respiration, ATP synthesis, and inorganic pyrophosphate (PPi) generation, and the mineralizing potential of released matrix vesicles (MV).


RESULTS
Articular chondrocytes and TC28 cells respired at comparable rates. Peroxynitrite and NO donors markedly suppressed respiration and ATP generation in chondrocytes. Because NO exerts multiple effects on chondrocytes, we investigated the primary functions of mitochondrial respiration and OXPHOS. To do so, we identified minimally cytotoxic doses of antimycin and oligomycin, which both induced intracellular ATP depletion (by 50-80%), attenuated collagen and proteoglycan synthesis, and blocked transforming growth factor beta from increasing intracellular ATP and elaboration of PPi, a critical inhibitor of hydroxyapatite deposition. Antimycin and oligomycin also abrogated the ability of the ATP-hydrolyzing enzyme plasma cell membrane glycoprotein 1 (PC-1) to increase chondrocyte PPi generation. Finally, MV from cells treated with antimycin or oligomycin contained less PPi and precipitated >50% more 45Ca.


CONCLUSIONS
Chondrocyte mitochondrial reserve, as NO-sensitive mitochondrial respiration-mediated ATP production, appears to support matrix synthesis and PPi elaboration and to regulate MV composition and mineralizing activity. NO-induced depression of chondrocyte respiration could modulate matrix loss and secondary cartilage mineralization in OA.
Johnson
K
K
Department of Veterans Affairs Medical Center, and University of California, San Diego 92161, USA.
Jung
A
A
Murphy
A
A
Andreyev
A
A
Dykens
J
J
Terkeltaub
R
R
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000968:Antimycin A / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002358:Cartilage, Articular / Q000166:cytology; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D011756:Diphosphates; D009840:Oligomycins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D009569:Nitric Oxide; D000968:Antimycin A; D000255:Adenosine Triphosphate