TNF-ALPHA INCREASES EXPRESSION OF IL-6 AND ICAM-1 GENES THROUGH ACTIVATION OF NF-KAPPA-B IN OSTEOBLAST-LIKE ROS17 2.8 CELLS/

Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammato ry diseases such as rheumatoid arthritis and in postmenopausal osteopo rosis, In various tissues, TNF-alpha action is mediated by a transcrip tion factor, nuclear factor-kappa B (NF-kappa B). However, little is k nown about how TNF-alpha exerts its action in osteoblasts, We thus exa mined the effect of TNF-alpha on the activation of NF-kappa B in rat o steoblast-like osteosarcoma cells (ROS17/2.8), Electrophoretic mobilit y shift assay revealed that the activation of the p50-p65 heterodimer NF-kappa B was induced by TNF-alpha as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-kap pa B in cytosol was examined using detergents that dissociate NF-kappa B from an inhibitory protein I kappa B, it decreased during the initi al 30 minutes and then increased to the unstimulated level. Northern b lot 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 mR NA levels during the initial 1 h. Significant increase in both mRNA le vels continued until 24 h after TNF-alpha. These results suggest that the rapid activation of NF-kappa B by TNF-alpha is mainly due to the n uclear translocation of NF-kappa B pre-existing in cytosol, and that t he subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-KB during TNF-alpha stimulation, TNF-a lpha also increased the mRNA levels of interleukin-6 (IL-6) and interc ellular adhesion molecule-1 (ICAM-1). An antioxidant, N-acetyl-L-cyste ine, significantly attenuated the TNF-alpha-dependent increase in thes e mRNAs, and simultaneously reduced the activation of NF-kappa B by TN F-alpha, indicating that NF-kappa B mediates the TNF-alpha-dependent e xpression of IL-6 and ICAM-1. in ROS17/2,8 cells. These results sugges t that the activation of NF-kappa B 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 inflammat ion.