Effect of a cyclooxygenase-2 inhibitor on interleukin-1β-stimulated activation of the transcription factor nuclear factor-kappa B in human gingival fibroblasts

Background: In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1 beta-stimulated prostaglandin E-2 (PGE(2)) production almost completely while partially inhibiting IL-6 production in aggressive periodontitis (AgP) human gingival fibroblasts. PGE(2) and the transcription factor nuclear factor-kappa B (NF-kappa B) regulate IL-1 beta-stimulated IL-6 production. Cytoplasmic NF-kappa B is boundto inhibitors (I kappa B proteins). IL-1 beta initiates a cascade resulting in phosphorylation and degradation of I kappa B, allowing nuclear translocation of NF-KB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of I kappa B and NF-kappa B activation. Methods: AgP fibroblasts (1 to 2 x 10(6)) were exposed to, IL-1 beta (1 x 10(-11) M) with or without NS-398 (10 nM) in serum-free medium. The NF-KB subunit p65 and phospho-I kappa B alpha were measured in whole cell, cytoplasmic, or nuclear extracts, using colorimetric assays. Enzyme-linked immunosorbent assays were used to measure PGE(2) and IL-6 production by 2.5 x 10(4) cells after exposure to IL-1 beta with or without NS-398 in serum-free medium. Results: Consistent with previous results, NS-398 reduced IL-1 beta-stimulated PGE(2) by similar to 98% (P < 0.001) and IL-6 by similar to 65% (P < 0.001). IL-1 beta increased nuclear and cytoplasmic p65 (similar to 8fold [P < 0.001] and similar to 2.5-fold [P < 0.03], respectively) over control levels. NS-398 reduced IL-1 beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1 beta increased phospho-I kappa B alpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P <= 0.008). Conclusions: NS-398 inhibited NF-KB activation and nuclear p65 levels in human gingival fibroblasts. This seemed to be due to inhibition of the phosphorylation cascade resulting in formation of phospho-I kappa B alpha and free p65. NF-KB inhibition may be useful in treating inflammatory diseases such as AgP.