Fushimi K, Nakashima S, Banno Y, Akaike A, Takigawa M, Shimizu K
Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan.
Osteoarthritis Cartilage. 2004 Nov;12(11):895-903. doi: 10.1016/j.joca.2004.08.001.
Calpains are known as Ca(2+)-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined.
Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined.
TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha.
These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.
钙蛋白酶是一种依赖钙离子的细胞内中性半胱氨酸蛋白酶。然而，在关节炎关节的滑液中检测到了m-钙蛋白酶，并且在体外显示其具有蛋白聚糖酶活性。关节炎期间m-钙蛋白酶释放到细胞外空间的机制尚未得到充分阐明。在本研究中，我们分析了促炎细胞因子肿瘤坏死因子-α（TNF-α）刺激培养的软骨细胞后m-钙蛋白酶的释放情况。还研究了非甾体抗炎药（NSAIDs）对m-钙蛋白酶释放的影响。
在有或没有NSAID的情况下，用TNF-α刺激人软骨细胞系HCS-2/8细胞。使用抗m-钙蛋白酶抗体通过蛋白质印迹分析对细胞和培养基中的m-钙蛋白酶进行定量。对蛋白质印迹进行光密度分析并测定条带强度。
TNF-α（10 ng/ml）刺激HCS-2/8细胞中m-钙蛋白酶的释放，并使细胞内m-钙蛋白酶短暂增加。所检测的NSAIDs（阿司匹林、氯索洛芬-SRS、双氯芬酸钠、吲哚美辛和NS398）抑制了10 ng/ml TNF-α诱导的m-钙蛋白酶释放和前列腺素E2（PGE2）的产生。外源性添加的PGE2加速了对较低浓度TNF-α（1 ng/ml）的反应中m-钙蛋白酶的释放。EP1/2拮抗剂AH6809有效抑制了TNF-α诱导的m-钙蛋白酶释放，而EP1拮抗剂SC19220则没有。相反，EP2激动剂布他前列素加速了1 ng/ml TNF-α诱导的m-钙蛋白酶释放。
这些结果表明，TNF-α刺激软骨细胞系HCS-2/8细胞中m-钙蛋白酶的上调和释放，并且产生的PGE2对EP2-PGE2受体的刺激在此过程中起重要作用。
