Oxidative stress reduces histone deacetylase 2 activity and enhances IL-8 gene expression: role of tyrosine nitration

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Abstract

Oxidative stress is a characteristic of chronic inflammatory diseases. The reactive oxygen intermediate hydrogen peroxide (H2O2) is an important signaling molecule that modulates gene expression. We have demonstrated that H2O2 significantly enhanced cytokine production in BEAS-2B cells, with a maximal effect at 4 h. This did not result from enhanced NF-κB activation, but through decreased activity of histone deacetylase (HDAC)2. This results in increased inflammatory gene expression following acetylation of specific histone residues. Decreased HDAC2 activity was associated with tyrosine nitration status. Peroxynitrite and SIN-1, a peroxynitrite generator, were also able to reduce HDAC2 activity via tyrosine nitration. Our data suggest that oxidative stress contributes to worsening inflammation via reduction of HDAC2 activity through HDAC2 nitration. This novel mechanism of inflammation may be important in increasing the severity and chronicity of inflammatory diseases.

Section snippets

Materials and methods

Materials. Thirty percentage of H2O2 and N-acetyl-l-cysteine (NAC) were purchased from Sigma (Sigma, Poole, UK); IL-1β was from R&D (Abingdon, UK); DCFH-DA and SIN-1 were from Molecular Probes (Leiden, Netherlands); and peroxynitrite was from Cayman Chemicals (Ann Arbor, MI, USA). Anti-p65 (sc-109, sc-7151), anti-phosphotyrosine (sc-508), anti-HDAC2 (sc-7899), and anti-HDAC1 (sc-6298) antibodies were obtained from Santa Cruz Biotec (Santa Cruz, California, USA), and anti-nitrotyrosine (1A6) was

H2O2 enhances IL-1β-stimulated cytokine expression

Pretreatment of human airway epithelial cells (BEAS2B) with H2O2 (100 μM) for 4 h slightly enhanced basal IL-8 production but markedly potentiated IL-1β-stimulated IL-8 production (2497 ± 226 ng/ml versus 1066 ± 64) (Fig. 1A) without affecting cell survival (data not shown). This enhancement by pretreatment of H2O2 was maximal at 4 h (Fig. 1B). Four hour pretreatment of H2O2 also enhanced GM-CSF production as well as IL-8 (211 ± 22 vs 138 ± 36). This effect was blocked by the anti-oxidant N-acetyl-l

Discussion

In this study we have demonstrated that H2O2 and peroxynitrite enhanced IL-1β-induced expression of inflammatory cytokines, such as GM-CSF and IL-8. This effect could be blocked by the anti-oxidant NAC. Using chromatin immunoprecipitation assays in cells treated with IL-1β plus H2O2, the IL-8 promoter region was associated with much greater levels of acetylated histone H4 than those after IL-1β stimulation alone. Under the same conditions, there was no change in p65-associated IL-8 promoter

Acknowledgements

This work was funded by the British Lung Foundation, the Clinical Research Committee (Royal Brompton Hospital), and GlaxoSmithKline plc (UK).

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