The role of adenosine in chondrocyte death in murine osteoarthritis and in a murine chondrocyte cell line.
OBJECTIVE
To investigate the role of adenosine in chondrocyte death in murine osteoarthritis (OA).


METHODS
5'-Nucleotidase (5'NT) generates adenosine. Enzyme activity was measured histochemically in normal murine and osteoarthritic STR/ort strain tibial cartilage. Adenosine-mediated cell death was investigated in MC615 chondrocyte cultures. Adenosine receptors (ARs) were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Cellular uptake of [(3)H] adenosine was measured with or without the inhibitor, nitrobenzylthioinosine (NBTI). Cell death was assessed by cell counting and DNA laddering following selective receptor stimulation, or after modulating adenosine metabolism with adenosine deaminase (ADA) or adenosine kinase (AK) inhibitors [erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and Iodotubericidin (Itub)], or with homocysteine (HC). Markers of apoptosis were assessed by Western blotting. Cell studies were validated by incubating normal murine knee joints in a medium containing adenosine and metabolic inhibitors. Apoptotic chondrocytes were identified with the TUNEL reaction.


RESULTS
5'NT activity in STR/ort tibial cartilage increased with development of OA, especially close to OA lesions. Adenosine induced MC615 cell death in the presence of ADA inhibition (100 microM EHNA), or 1mM HC, or both. Adenosine uptake, mediated by NBTI-sensitive adenosine transporters, was required for cell death. ARs were expressed (A2b>A2a>A1) but were not involved in mediating cell death. Cell death involved the activation of caspase-3 and DNA fragmentation and was prevented by inhibiting caspase activity. However, neither caspase-8 nor caspase-9 was detected. Adenosine+EHNA induced chondrocyte apoptosis in normal murine knee joints.


CONCLUSIONS
Increased adenosine production may induce chondrocyte apoptosis and play a role in OA in STR/ort mice.
Mistry
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Department of Clinical Pharmacology, William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Chambers
M G
MG
Mason
R M
RM
D015720:5'-Nucleotidase / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000241:Adenosine / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors; D000248:Adenosine Kinase / Q000037:antagonists & inhibitors; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002358:Cartilage, Articular / Q000378:metabolism / Q000503:physiopathology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D053938:DNA Fragmentation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006710:Homocysteine / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010003:Osteoarthritis / Q000503:physiopathology*; D018047:Receptors, Purinergic P1 / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014372:Tubercidin / Q000031:analogs & derivatives / Q000494:pharmacology
D058892:Adenosine Deaminase Inhibitors; D004791:Enzyme Inhibitors; D018047:Receptors, Purinergic P1; D006710:Homocysteine; C010192:9-(2-hydroxy-3-nonyl)adenine; D000248:Adenosine Kinase; D015720:5'-Nucleotidase; D000225:Adenine; D000241:Adenosine; D014372:Tubercidin