Annexin 5 overexpression increased articular chondrocyte apoptosis induced by basic calcium phosphate crystals.
OBJECTIVE
Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis. In advanced osteoarthritis, cartilage shows chondrocyte apoptosis, overexpression of annexin 5 (A5) and BCP crystal deposition within matrix vesicles. We assessed in vitro whether BCP crystals and overexpression of A5 increased chondrocyte apoptosis.


METHODS
Apoptosis was induced by BCP crystals, tumour necrosis factor (TNF)-alpha (20 ng/ml) and Fas ligand (20 ng/ml) in normal articular chondrocytes (control) and in A5 overexpressed chondrocytes, performed by adenovirus infection. Apoptosis was assessed by caspase 3 (Cas3) activity, and DNA fragmentation.


RESULTS
All BCP crystals, TNF-alpha and Fas ligand induced chondrocyte apoptosis as demonstrated by decreased cell viability and increased Cas3 activity and DNA fragmentation. TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end-labelling)-positive staining chondrocytes were increased by OCP (12.4 (5.2)%), CA (9.6 (2.6)%) and HA (9.2 (3.0)%) crystals and TNF-alpha (9.6 (2.4)%) stimulation compared with control (3.1 (1.9)%). BCP crystals increased Cas3 activity in a dose-dependent fashion. BCP-crystal-induced chondrocyte apoptosis was independent from TNF-alpha and interleukin-1beta pathways but required cell-crystal contact and intralysosomal crystal dissolution. Indeed, preincubation with ammonium chloride, a lysosomal inhibitor of BCP crystal dissolution, significantly decreased BCP-crystal-induced Cas3 activity. Finally, overexpression of A5 enhanced BCP crystal- and TNF-alpha-induced chondrocyte apoptosis.


CONCLUSIONS
Overexpression of A5 and the presence of BCP crystals observed in advanced osteoarthritis contributed to chondrocyte apoptosis. Our results suggest a new pathophysiological mechanism for calcium-containing crystal arthropathies.
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INSERM U606, IFR 139, Hôpital Lariboisière, Paris, France.
Monceau
V
V
Camors
E
E
Cohen-Solal
M
M
Charlemagne
D
D
Lioté
F
F
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism / Q000502:physiology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002130:Calcium Phosphates / Q000378:metabolism / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D003460:Crystallization; D053938:DNA Fragmentation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D014527:Uric Acid / Q000378:metabolism
D017304:Annexin A5; D002130:Calcium Phosphates; D014409:Tumor Necrosis Factor-alpha; D014527:Uric Acid; D053148:Caspase 3