title: Oestrogens inhibit interleukin 1beta-mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor-kappa B impairment.
abstract: OBJECTIVE
  To investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression.
  
  
  METHODS
  The presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry.
  
  
  RESULTS
  ERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2.
  
  
  CONCLUSIONS
  Our data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.
authors.affiliation: INSERM UMR-747, Universite Paris Descartes, UFR Biomedicale, Paris, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Richette
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Dumontier
authors.forename: Khadija
authors.initials: K
authors.lastname: Tahiri
authors.forename: Magdalena
authors.initials: M
authors.lastname: Widerak
authors.forename: Antoine
authors.initials: A
authors.lastname: Torre
authors.forename: Mourad
authors.initials: M
authors.lastname: Benallaoua
authors.forename: Mourad
authors.initials: M
authors.lastname: Benallaloua
authors.forename: François
authors.initials: F
authors.lastname: Rannou
authors.forename: Marie-Therese
authors.initials: MT
authors.lastname: Corvol
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Savouret
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000378:metabolism / Q000502:physiology*; D005260:Female; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D014178:Translocation, Genetic / Q000187:drug effects
chemical_list: D047628:Estrogen Receptor alpha; D053583:Interleukin-1beta; D016328:NF-kappa B; D009569:Nitric Oxide; D004958:Estradiol; D019001:Nitric Oxide Synthase