Ea H K, Monceau V, Camors E, Cohen-Solal M, Charlemagne D, Lioté F
INSERM U606, IFR 139, Hôpital Lariboisière, Paris, France.
Ann Rheum Dis. 2008 Nov;67(11):1617-25. doi: 10.1136/ard.2008.087718. Epub 2008 Jan 24.
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.
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.
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.
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.
碱性磷酸钙（BCP）晶体（八钙磷酸酯（OCP）、碳磷灰石（CA）和羟基磷灰石（HA））与严重形式的骨关节炎相关。在晚期骨关节炎中，软骨表现出软骨细胞凋亡、膜联蛋白5（A5）过表达以及基质小泡内BCP晶体沉积。我们在体外评估了BCP晶体和A5过表达是否会增加软骨细胞凋亡。
通过腺病毒感染，在正常关节软骨细胞（对照）和A5过表达的软骨细胞中，用BCP晶体、肿瘤坏死因子（TNF）-α（20 ng/ml）和Fas配体（20 ng/ml）诱导凋亡。通过半胱天冬酶3（Cas3）活性和DNA片段化评估凋亡情况。
所有BCP晶体、TNF-α和Fas配体均诱导软骨细胞凋亡，表现为细胞活力降低、Cas3活性增加和DNA片段化。与对照（3.1（1.9）%）相比，OCP（12.4（5.2）%）、CA（9.6（2.6）%）和HA（9.2（3.0）%）晶体以及TNF-α（9.6（2.4）%）刺激后，TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）阳性染色的软骨细胞增加。BCP晶体以剂量依赖性方式增加Cas3活性。BCP晶体诱导的软骨细胞凋亡独立于TNF-α和白细胞介素-1β途径，但需要细胞与晶体接触以及溶酶体内晶体溶解。实际上，用氯化铵（一种BCP晶体溶解的溶酶体抑制剂）预孵育可显著降低BCP晶体诱导的Cas3活性。最后，A5过表达增强了BCP晶体和TNF-α诱导的软骨细胞凋亡。
晚期骨关节炎中观察到的A5过表达和BCP晶体的存在促成了软骨细胞凋亡。我们的结果提示了含钙晶体关节病的一种新的病理生理机制。
