Clancy R, Rediske J, Koehne C, Stoyanovsky D, Amin A, Attur M, Iyama K, Abramson S B
Department of Rheumatology, Hospital for Joint Diseases and Division of Medicine, NYU School of Medicine, New York, New York, 10003, USA.
Osteoarthritis Cartilage. 2001 May;9(4):294-9. doi: 10.1053/joca.2000.0388.
We have demonstrated in bovine chondrocytes that nitric oxide (NO) mediates IL1 dependent apoptosis under conditions of oxidant stress. This process is accompanied by activation of c-Jun NH2-terminal kinase (JNK; also called stress-activated protein kinase). In these studies we examined activation of JNK in explant cultures of human osteoarthritic cartilage obtained at joint replacement surgery and we characterized the role of peroxynitrite to act as an upstream trigger.
A novel technique to isolate chondrocyte proteins (<10% of total cartilage protein) from cartilage specimens was developed. It was used to analyse JNK activation by a western blot technique. To examine the hypothesis that chondrocyte JNK activation is a result of increased peroxynitrite, in vitro experiments were performed in which cultured chondrocytes were incubated with this oxidant.
Activated JNK was detected in the cytoplasm of osteoarthritis (OA) affected chondrocytes but not in that of controls. In vitro, chondrocytes produce NO and superoxide anion. IL-1 (48 h), which induces nitric oxide synthase, resulted in an activation of JNK; this effect was reversed by N-monomethylarginine (NMA). TNFalpha treated chondrocytes at 48 h produce superoxide anion (EPR method). Exposure of cells to peroxynitrite led to an accumulation of intracellular oxidants, in association with JNK activation and cell death by apoptosis.
We suggest that JNK activation is among the IL-1 elicited responses that injure articular chondrocytes and this activation of JNK is dependent on intracellular oxidant formation (including NO peroxynitrite). In addition, the extraction technique here described is a novel method that permits the quantitation and study of proteins such as JNK involved in the signaling pathways of chondrocytes within osteoarthritic cartilage.
我们已在牛软骨细胞中证实，在氧化应激条件下，一氧化氮（NO）介导白细胞介素-1（IL-1）依赖性细胞凋亡。这一过程伴随着c-Jun氨基末端激酶（JNK；也称为应激激活蛋白激酶）的激活。在这些研究中，我们检测了在关节置换手术中获取的人骨关节炎软骨外植体培养物中JNK的激活情况，并确定了过氧亚硝酸盐作为上游触发因素的作用。
开发了一种从软骨标本中分离软骨细胞蛋白（占总软骨蛋白的<10%）的新技术。该技术用于通过蛋白质印迹技术分析JNK的激活情况。为检验软骨细胞JNK激活是过氧亚硝酸盐增加所致这一假说，进行了体外实验，将培养的软骨细胞与这种氧化剂一起孵育。
在骨关节炎（OA）受累软骨细胞的细胞质中检测到活化的JNK，而在对照细胞中未检测到。在体外，软骨细胞产生NO和超氧阴离子。诱导一氧化氮合酶的IL-1（48小时）导致JNK激活；N-单甲基精氨酸（NMA）可逆转这一效应。肿瘤坏死因子α（TNFα）处理48小时的软骨细胞产生超氧阴离子（电子顺磁共振方法）。细胞暴露于过氧亚硝酸盐会导致细胞内氧化剂积累，同时伴有JNK激活和细胞凋亡死亡。
我们认为JNK激活是IL-1引发的损伤关节软骨细胞的反应之一，且这种JNK激活依赖于细胞内氧化剂的形成（包括NO和过氧亚硝酸盐）。此外，本文所述的提取技术是一种新方法，可对参与骨关节炎软骨中软骨细胞信号通路的蛋白质（如JNK）进行定量和研究。
