Oestrogens inhibit interleukin 1β-mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor-κB impairment

Objectives: To investigate the presence and functionality of oestrogen receptor alpha (ER alpha) in interleukin (IL) 1 beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17 beta oestradiol (E2) effects on IL1 beta-induced inducible nitric oxide synthase (iNOS) expression. Methods: The presence and functionality of ER alpha 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-kappa B (NF-kappa B) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry. Results: ER alpha was identified in the nucleus of chondrocytes. ERa efficiently transactivated a transiently expressed E2-responsive construct. On IL1 beta treatment, ERa partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1 beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1 beta-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 IL1 beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1 beta was also strongly decreased by E2. Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1 beta, 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.