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J Periodontol. 2007 Mar;78(3):542-9.

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

Author information

1
Dental Research Center and Department of Periodontology, College of Dentistry, The University of Tennessee Health Science Center, Memphis, TN 38163, USA. dtipton@utmem.edu

Abstract

BACKGROUND:

In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1beta-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-kappaB) regulate IL-1beta-stimulated IL-6 production. Cytoplasmic NF-kappaB is bound to inhibitors (IkappaB proteins). IL-1beta initiates a cascade resulting in phosphorylation and degradation of IkappaB, allowing nuclear translocation of NF-kappaB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of IkappaB and NF-kappaB activation.

METHODS:

AgP fibroblasts (1 to 2 x 10(6)) were exposed to IL-1beta (1 x 10(11)M) with or without NS-398 (10 nM) in serum-free medium. The NF-kappaB subunit p65 and phospho-IkappaBalpha 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-1beta with or without NS-398 in serum-free medium.

RESULTS:

Consistent with previous results, NS-398 reduced IL-1beta-stimulated PGE(2) by approximately 98% (P <0.001) and IL-6 by approximately 65% (P <0.001). IL-1beta increased nuclear and cytoplasmic p65 ( approximately 8-fold [P <0.001] and approximately 2.5-fold [P <0.03], respectively) over control levels. NS-398 reduced IL-1beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1beta increased phospho-IkappaBalpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P <or=0.008).

CONCLUSIONS:

NS-398 inhibited NF-kappaB 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-IkappaBalpha and free p65. NF-kappaB inhibition may be useful in treating inflammatory diseases such as AgP.

PMID:
17335379
DOI:
10.1902/jop.2007.060250
[Indexed for MEDLINE]

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