Kurokouchi K, Kambe F, Yasukawa K, Izumi R, Ishiguro N, Iwata H, Seo H
Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Japan.
J Bone Miner Res. 1998 Aug;13(8):1290-9. doi: 10.1359/jbmr.1998.13.8.1290.
Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF-alpha action is mediated by a transcription factor, nuclear factor-kappa B (NF-kappaB). However, little is known about how TNF-alpha exerts its action in osteoblasts. We thus examined the effect of TNF-alpha on the activation of NF-kappaB in rat osteoblast-like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50-p65 heterodimer NF-kappaB was induced by TNF-alpha as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-kappaB in cytosol was examined using detergents that dissociate NF-kappaB from an inhibitory protein IkappaB, it decreased during the initial 30 minutes and then increased to the unstimulated level. Northern blot analysis revealed a marked increase in the mRNA levels of p105, a precursor of p50, 6 h after TNF-alpha and a gradual increase in p65 mRNA levels during the initial 1 h. Significant increase in both mRNA levels continued until 24 h after TNF-alpha. These results suggest that the rapid activation of NF-kappaB by TNF-alpha is mainly due to the nuclear translocation of NF-kappaB pre-existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-kappaB during TNF-alpha stimulation. TNF-alpha also increased the mRNA levels of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1). An antioxidant, N-acetyl-L-cysteine, significantly attenuated the TNF-alpha-dependent increase in these mRNAs, and simultaneously reduced the activation of NF-kappaB by TNF-alpha, indicating that NF-kappaB mediates the TNF-alpha-dependent expression of IL-6 and ICAM-1 in ROS17/2.8 cells. These results suggest that the activation of NF-kappaB by TNF-alpha may play an important role in the production of cytokines and cell adhesion molecules from osteoblasts, leading to the promotion of bone resorption and inflammation.
肿瘤坏死因子-α（TNF-α）在类风湿性关节炎等炎症性疾病以及绝经后骨质疏松症中起关键作用。在各种组织中，TNF-α的作用是由转录因子核因子-κB（NF-κB）介导的。然而，关于TNF-α如何在成骨细胞中发挥作用却知之甚少。因此，我们研究了TNF-α对大鼠成骨样骨肉瘤细胞（ROS17/2.8）中NF-κB激活的影响。电泳迁移率变动分析显示，TNF-α早在15分钟就诱导了p50-p65异二聚体NF-κB的激活，随后持续激活48小时。当使用能使NF-κB与抑制蛋白IκB解离的去污剂检测细胞质中NF-κB的结合活性时，其在最初30分钟内降低，然后升高至未刺激水平。Northern印迹分析显示，TNF-α作用6小时后，p50的前体p105的mRNA水平显著增加，在最初1小时内p65的mRNA水平逐渐增加。两种mRNA水平的显著增加一直持续到TNF-α作用24小时。这些结果表明，TNF-α对NF-κB的快速激活主要是由于细胞质中预先存在的NF-κB的核转位，并且随后p50和p65表达的增加可能导致TNF-α刺激期间NF-κB的持续激活。TNF-α还增加了白细胞介素-6（IL-6）和细胞间黏附分子-1（ICAM-1）的mRNA水平。抗氧化剂N-乙酰-L-半胱氨酸显著减弱了这些mRNA的TNF-α依赖性增加，同时降低了TNF-α对NF-κB的激活，表明NF-κB介导了ROS17/2.8细胞中IL-6和ICAM-1的TNF-α依赖性表达。这些结果表明，TNF-α对NF-κB的激活可能在成骨细胞产生细胞因子和细胞黏附分子中起重要作用，从而导致骨吸收和炎症的促进。
