Objectives: To investigate the presence and functionality of oestrogen receptor α (ERα) in interleukin (IL)1β-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17β oestradiol (E2) effects on IL1β-induced inducible nitric oxide synthase (iNOS) expression.
Methods: The presence and functionality of ERα 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-κB (NF-κB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)–ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry.
Results: ERα was identified in the nucleus of chondrocytes. ERα efficiently transactivated a transiently expressed E2-responsive construct. On IL1β treatment, ERα partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1β-induced NO production (−34%, −31% and −36%, respectively). E2 decreased IL1β-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 IL1β-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1β was also strongly decreased by E2.
Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1β, 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.
- Ap-1, actovated protein 1
- ERα, oestrogen receptor α
- ERE, oestrogen responsive element
- ERT, oestrogen replacement therapy
- iNOS, inducible nitric oxide synthase
- NF-κB, nuclear factor κB
- SERM, selective oestrogen receptor modulators
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Published Online First 1 November 2006
Competing interests: None.