Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor α, and inhibition by glucocorticoids

Abstract

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 1beta (IL-1beta) and tumour necrosis factor alpha, 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-1beta (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 kappaB (NFkappaB)-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-1beta (10 pM)-induced IkBalpha degradation, NFkappaB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFkappaB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBalpha gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFkappaB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.

Anti-inflammatory functions of glucocorticoid-induced genes

“…In some studies anti-inflammatory effects of GCs were not accompanied by changes in DNA-binding activity of NFκB as measured by electrophoretic mobility shift assay (Brostjan et al, 1996;De Bosscher et al, 1997;Hofmann et al, 1998;Nissen and Yamamoto, 2000;Ray et al, 1997;Wissink et al, 1998). However, in other cases GCs were found to inhibit NFκB activation as measured by the same assay (Eberhardt et al, 2002;Goppelt-Struebe et al, 2000;Kurata and Yamamoto, 1999;Kurokouchi et al, 2000;Ma et al, 2004;Mukaida et al, 1994;Vital et al, 2003).…”

“…A number of problems with this hypothesis have since been raised. In several different cell types the GC-induced upregulation of IκBα either did not occur or was not sufficient to alter the degradation of IκBα protein, and the nuclear translocation of NFκB in response to pro-inflammatory stimuli (Adcock et al, 1999;Adcock et al, 1997;Auwardt et al, 1998;Brostjan et al, 1996;De Bosscher et al, 1997;Hofmann et al, 1998;Liden et al, 2000;Nissen and Yamamoto, 2000;Pruett et al, 2003;Ray et al, 1997;Wissink et al, 1998). As described above, inhibition of inflammatory gene expression could occur without apparent changes in NFκB promoter occupancy (Liden et al, 2000;Luecke and Yamamoto, 2005 All of these observations clearly establish that inhibition of NFκB function can occur independently of IκBα induction.…”

Separating Transrepression and Transactivation: A Distressing Divorce for the Glucocorticoid Receptor?

“…However, the induction of IB␣ is not necessary for repression of NF-B-dependent transcription in many cells (Heck et al, 1997;Wissink et al, 1998). For example, in pulmonary type II and endothelial cells stimulated with tumor necrosis factor ␣, IL-1␤, or lipopolysaccharide, IB␣ expression was not increased, and NF-B DNA binding was unaltered over time frames in which glucocorticoid-dependent repression of gene expression occurred (Brostjan et al, 1996;Ray et al, 1997;Newton et al, 1998a).…”

Carbohydrate‐mediated cell adhesion in cancer metastasis and angiogenesis

“…There are many other factors that lead to an increment of vascular E-selectin expression, such as surgical operation, chemotherapy, radiotherapy, or opportunistic infections, while glucocorticoids suppress its expression. 19,20) …”