The effect of oxidative stress and glucocorticoids on TGF-ß1 production in airway epithelial cells

Transforming growth factor-β1 (TGF-β1) is implicated in inflammation and airway wall remodeling in inflammatory airway disease. Oxidative stress has also been implicated in the pathogenesis of inflammatory lung disorders. Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), in the airway may activate intracellular signalling cascades and transcription factors to exaggerate lung inflammation. The balance between nuclear histone acetylation/deacetylation has also been shown to be altered by oxidative stress and may further upregulate lung inflammation. The aim of this thesis was to determine whether ROS, alone or in combination with interleukin (IL)-1β can modulate TGF-β1 expression in lung epithelial cells and whether the effect was regulated by histone methylation. Stimulation of human airway epithelial cells (A549) with H2O2 or H2O2 plus IL-1β enhanced TGF-β1 mRNA and total protein expression. IL-1β and H2O2 together showed a greater than additive effect on TGF-β1 mRNA transcription, but not on the total protein level. The NF-[kappa]B-IKK2 inhibitor AS602868 suppressed IL-1β-and H2O2 plus IL-1β -induced TGF- β1 mRNA expression. NF-[Kappa]B p65 protein was also shown to be upregulated by IL-1β plus H2O2 stimulation. Chromatin immunoprecipitation (ChIP) experiments demonstrated that p65 was recruited to the [Kappa]B3 binding site on the native TGF-β1 promoter area. The upregulation of TGF-β1 mRNA by IL-β plus H2O2 could be suppressed by fluticasone propionate (FP) although to the same extent as seen with IL-1β-stimulated TGF-β1 mRNA expression. This suggests that H2O2 induced a relative steroid insensitivity. Both Suv39H1 protein level and mRNA expression were downregulated by IL-1β and H2O2 stimulation. Knockdown of Suv39H1 with siRNA transfection induced a trend towards increased TGF-ß1 mRNA expression upon stimulation with IL-1β and H2O2. Suv39H1 was shown to be recruited to glucocorticoid receptor by co-immunoprecipitation. The anti-inflammatory effect of FP was impaired under Suv39H1 knockdown which indicate that Suv39H1 may act as a corepressor of GR function. DNA methylation is also involved in epigenetic regulation of gene expression. IL-1β and H2O2 stimulation rapidly upregulated CpG methylation at specific sites in the TGF-β1 promoter as demonstrated by direct sequencing after bisulphite treatment and by methylation dependent immunoprecipitation study (MeDIP). Methylation at the ATG start start induced by IL-1β was reversed over time indicating an active demethylation. These results indicate that H2O2, acting via the NF-[Kappa]B pathway, increases TGF-ß1 mRNA production and that this is regulated by FP possibly through an effect on the histone methyltransferase, Suv39H1. DNA methylation is also involved in the regulation of IL-1β plus H2O2 induced TGF-β1 mRNA expression