Suppression of Granulocyte-Macrophage Colony-Stimulating Factor Expression by Glucocorticoids Involves Inhibition of Enhancer Function by the Glucocorticoid Receptor Binding to Composite NF-AT/Activator Protein-1 Elements

Abstract

Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.

Reversing the defective induction of IL-10-secreting regulatory T cells in glucocorticoid-resistant asthma patients

“…CD4 + T cells were incubated with human recombinant IL-10 (R&D Systems), vitamin D3, and/or dexamethasone for the indicated periods (0 to 72 hours), then they were washed, soluble cell proteins were extracted, and Western blot was performed as previously described (56). GR was detected using a mouse monoclonal antibody (BD Transduction Laboratories; BD Biosciences -Pharmingen) and goat antimouse horseradish peroxidase-conjugated secondary antibody.…”

“…An empty vector (pcDNA3; Invitrogen Corp.) was used as a negative control. Overexpression of these cDNAs in U293 cells permitted Western blotting with either anti-FLAG (Sigma-Aldrich; not shown) or anti-GR (56). The different migration of these 2 proteins and comparison with native protein allowed discrimination among spliced variants.…”

Stress responses and innate immunity: aging as a contributory factor

“…A major anti-inflammatory mechanism of glucocorticoids such as cortisol is the suppression of pro-inflammatory cytokine synthesis and reduction of inflammatory mediators by inhibiting expression of enzymes such as cyclooxegenase-2 (COX-2) (Wallace et al ., 1997;Santini et al ., 2001). These effects are achieved by a variety of mechanisms, which include induction of transcriptional repressors by the cytosolic glucocorticoid receptor (Wallace et al ., 1997), and interaction of the cytosolic glucocorticoid receptor with transcription factors such as NF-κ B and AP-1, resulting in repression of their transcriptional activity (Reichardt et al ., 2001;Smith et al ., 2001;Ma et al ., 2004;Park et al ., 2004). Glucocorticoids can also prevent histamine release by inhibiting the degranulation of basophils, though their effect on mast cells is minimal.…”

Changes in angiotensin II type 1 receptor signalling pathways evoked by a monoclonal antibody raised to the N-terminus

“…After 12 h of transfection, cells were washed with PBS and then incubated in the absence or presence of Ang II at 10 K7 mol/l and/or purified 6313/G2, 2 . 5 mg/ml, in 2% FBS culture medium for 24 h. Luciferase assays were performed using a Luminoskan microplate luminometer and Ascent Software (ThermoLabsystems, Helsinki, Finland), and b-galactosidase was assayed using standard procedures (Smith et al 2001).…”