Huser C A M, Davies M E
University of Cambridge, Cambridge, UK.
Arthritis Rheum. 2007 Jul;56(7):2322-34. doi: 10.1002/art.22717.
Chondrocyte apoptosis is an important factor in the progression of osteoarthritis. This study aimed to elucidate the mechanisms involved upstream of caspase 9 activation and, in particular, calcium signaling and mitochondrial depolarization.
Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500-gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified by the TUNEL method. Release of proteoglycans was determined by the dimethylmethylene blue assay. Weight change was measured, and mitochondrial depolarization was determined using JC-1 staining. To assess the role of calcium signaling in impact-induced chondrocyte death, explants were preincubated in culture medium containing various concentrations of calcium. Inhibitors were used to assess the role of individual signaling components in impact-induced chondrocyte death.
Calcium quenching, inhibitors of calpains, calcium/calmodulin-regulated kinase II (CaMKII), and mitochondrial depolarization reduced impact-induced chondrocyte death after 48 hours in culture. Transient mitochondrial depolarization was observed 3-6 hours following a single impact load. Mitochondrial depolarization was prevented by calcium quenching, inhibitors of calpain, CaMKII, permeability transition pore formation, ryanodine receptor, and the mitochondrial uniport transporter. Cathepsin B did not appear to be involved in impact-induced chondrocyte death. The calpain inhibitor prevented proteoglycan loss, but the percentage weight gain and proteoglycan loss were unaffected by all treatments used.
Following a single impact load, calcium is released from the endoplasmic reticulum via the ryanodine receptor and is taken up by the mitochondria via the uniport transporter, causing mitochondrial depolarization and caspase 9 activation. In addition, calpains and CaMKII play important roles in causing mitochondrial depolarization.
软骨细胞凋亡是骨关节炎进展的一个重要因素。本研究旨在阐明半胱天冬酶9激活上游所涉及的机制，特别是钙信号和线粒体去极化。
从健康马获取关节软骨外植体，施加单次冲击负荷（500克重物从50毫米高度落下），并在体外培养长达48小时。通过TUNEL法对软骨细胞死亡进行定量。采用二甲基亚甲基蓝法测定蛋白聚糖的释放。测量重量变化，并使用JC-1染色测定线粒体去极化。为评估钙信号在冲击诱导的软骨细胞死亡中的作用，将外植体在含有不同浓度钙的培养基中预孵育。使用抑制剂评估各个信号成分在冲击诱导的软骨细胞死亡中的作用。
钙淬灭、钙蛋白酶抑制剂、钙/钙调蛋白调节激酶II（CaMKII）以及线粒体去极化减少了培养48小时后冲击诱导的软骨细胞死亡。单次冲击负荷后3 - 6小时观察到短暂的线粒体去极化。钙淬灭、钙蛋白酶抑制剂、CaMKII、通透性转换孔形成、兰尼碱受体和线粒体单向转运体的抑制剂可防止线粒体去极化。组织蛋白酶B似乎未参与冲击诱导的软骨细胞死亡。钙蛋白酶抑制剂可防止蛋白聚糖丢失，但所有处理均未影响重量增加百分比和蛋白聚糖丢失情况。
单次冲击负荷后，钙通过兰尼碱受体从内质网释放，并通过单向转运体被线粒体摄取，导致线粒体去极化和半胱天冬酶9激活。此外，钙蛋白酶和CaMKII在引起线粒体去极化中起重要作用。
