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Tumor necrosis factor-α (TNF-α) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF-α action is mediated by a transcription factor, nuclear factor-kappa B (NF-κB). However, little is known about how TNF-α exerts its action in osteoblasts. We thus examined the effect of TNF-α on the activation of NF-κB in rat osteoblast-like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50-p65 heterodimer NF-κB was induced by TNF-α as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-κB in cytosol was examined using detergents that dissociate NF-κB from an inhibitory protein IκB, 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-α 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-α. These results suggest that the rapid activation of NF-κB by TNF-α is mainly due to the nuclear translocation of NF-κB pre-existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-κB during TNF-α stimulation. TNF-α 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-α-dependent increase in these mRNAs, and simultaneously reduced the activation of NF-κB by TNF-α, indicating that NF-κB mediates the TNF-α-dependent expression of IL-6 and ICAM-1 in ROS17/2.8 cells. These results suggest that the activation of NF-κB by TNF-α 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.