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Effect of a Cyclooxygenase‐2 Inhibitor on Interleukin‐1β–Stimulated Activation of the Transcription Factor Nuclear Factor‐Kappa B in Human Gingival Fibroblasts
Author(s) -
Tipton David A.,
Gay Denise C.,
DeCoster Vaughn A.
Publication year - 2007
Publication title -
journal of periodontology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.036
H-Index - 156
eISSN - 1943-3670
pISSN - 0022-3492
DOI - 10.1902/jop.2007.060250
Subject(s) - microbiology and biotechnology , prostaglandin e2 , interleukin , chemistry , nfkb1 , transcription factor , cyclooxygenase , prostaglandin e , cytoplasm , phosphorylation , endocrinology , medicine , biology , enzyme , biochemistry , cytokine , gene
Background: In previous work, the cyclooxygenase‐2 inhibitor NS‐398 inhibited interleukin (IL)‐1β–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‐κB) regulate IL‐1β–stimulated IL‐6 production. Cytoplasmic NF‐κB is bound to inhibitors (IκB proteins). IL‐1β initiates a cascade resulting in phosphorylation and degradation of IκB, allowing nuclear translocation of NF‐κB and target gene activation. The purpose of this study was to determine whether NS‐398 inhibited phosphorylation of IκB and NF‐κB activation. Methods: AgP fibroblasts (1 to 2 × 10 6 ) were exposed to IL‐1β (1 × 10 −11 M) with or without NS‐398 (10 nM) in serum‐free medium. The NF‐κB subunit p6‐ and phospho‐IκBα 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.‐ × 10 4 cells after exposure to IL‐1β with or without NS‐398 in serum‐free medium. Results: Consistent with previous results, NS‐398 reduced IL‐1β–stimulated PGE 2 by ∼98% ( P <0.001) and IL‐6 by ∼65% ( P <0.001). IL‐1β increased nuclear and cytoplasmic p65 (∼8‐fold [ P <0.001] and ∼2.5‐fold [ P <0.03], respectively) over control levels. NS‐398 reduced IL‐1β–stimulated nuclear and cytoplasmic p65 to control levels. IL‐1β increased phospho‐IκBα 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‐κB 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‐IκBα and free p65. NF‐κB inhibition may be useful in treating inflammatory diseases such as AgP.