INDUCTION OF THE E-SELECTIN PROMOTER BY INTERLEUKIN-1 AND TUMOR-NECROSIS-FACTOR-ALPHA, AND INHIBITION BY GLUCOCORTICOIDS

Cytokine-induced expression of the endothelial cell surface adhesion m olecule E-selectin is inhibited by glucocorticoids (GCs). To investiga te possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-se lectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was hi ghly responsive to the cytokines interleukin 1 beta (IL-1 beta) and tu mour necrosis factor alpha, with ED50 values of 3 pM and 1000 pM respe ctively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppres sion of the E-selectin gene were mediated via regulatory elements with in the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1 beta (10 pM)-stimulated ESAP-gene activation we re fluticasone > beclomethasone > dexamethasone, with IC50 values of 0 .13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocke d by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via t he GR. However, because the E-selectin promoter lacks a consensus gluc ocorticoid responsive element, mechanisms for inhibition independent o f GR-DNA binding were investigated, Evidence that GCs also inhibited c ytokine activation of a synthetic nuclear factor kappa B (NF kappa B)- driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcr iption factor was important for GC inhibition of ESAP, However, in A54 9-ESAP cells, fluticasone (100 nM) did not affect IL-1 beta (10 pM)ind uced IkB alpha degradation, NF kappa B-p65 nuclear translocation or th e DNA-binding capacity of nuclear NF kappa B complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Fina lly, there was no evidence to suggest that GC enhancement of IkB alpha gene expression contributed to the suppression of the cytokine respon se. We conclude that interference by GR with the transcriptional activ ation potential of DNA-bound NF kappa B complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.