Cytokine-induced expression of the endothelial cell surface adhesion molecule E-selectin is inhibited by glucocorticoids (GCs). To investigate possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-selectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was highly responsive to the cytokines interleukin 1β (IL-1β) and tumour necrosis factor α, with ED50 values of 3 pM and 1000 pM respectively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppression of the E-selectin gene were mediated via regulatory elements within the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1β (10 pM)-stimulated ESAP-gene activation were fluticasone > beclomethasone > dexamethasone, with IC50 values of 0.13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocked by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via the GR. However, because the E-selectin promoter lacks a consensus glucocorticoid responsive element, mechanisms for inhibition independent of GR-DNA binding were investigated. Evidence that GCs also inhibited cytokine activation of a synthetic nuclear factor κB (NFκB)-driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcription factor was important for GC inhibition of ESAP. However, in A549-ESAP cells, fluticasone (100 nM) did not affect IL-1β (10 pM)-induced IkBα degradation, NFκB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFκB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBα gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFκB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.
Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor α, and inhibition by glucocorticoids
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P. Keith RAY, Stuart FARROW, Maria DALY, Francois TALABOT, Nicola SEARLE; Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor α, and inhibition by glucocorticoids. Biochem J 1 December 1997; 328 (2): 707–715. doi: https://doi.org/10.1042/bj3280707
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