title: Effect of a cyclooxygenase-2 inhibitor on interleukin-1beta-stimulated activation of the transcription factor nuclear factor-kappa B in human gingival fibroblasts.
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.
authors.affiliation: Dental Research Center and Department of Periodontology, College of Dentistry, The University of Tennessee Health Science Center, Memphis, TN 38163, USA. dtipton@utmem.edu
authors.forename: David A
authors.initials: DA
authors.lastname: Tipton
authors.forename: Denise C
authors.initials: DC
authors.lastname: Gay
authors.forename: Vaughn A
authors.initials: VA
authors.lastname: DeCoster
mesh_terms: D000293:Adolescent; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D015232:Dinoprostone / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009578:Nitrobenzenes / Q000494:pharmacology*; D010518:Periodontitis / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D020672:I-kappa B Proteins; D053583:Interleukin-1beta; D015850:Interleukin-6; D016328:NF-kappa B; D009578:Nitrobenzenes; D013449:Sulfonamides; D051996:Transcription Factor RelA; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D015232:Dinoprostone