Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation.

Inhaled glucocorticosteroids (GCs) are the most effective therapy for the control of asthma and are now advocated as first line therapy for most patients. Steroids reduce airway hyper-responsiveness and suppress the airway inflammatory response in asthma, but their exact mechanisms and cellular targets in the lung are uncertain 111. Classically GCs bind to a cytosolic 94kDa receptor protein, that is held in an inactive, non-DNA binding state by a 9OkDa heat shock protein (hsp90). With lioand binding, the DNA binding site is revealed and enhancement of the transcriptional response occurs following the translocation of active receptor to the nucleus. The GR binds to a consensus cis-acting DNA sequence AGAACAnnnTGTTCT (GRE) in the 5’ upstream controlling region of the genes under positive control by GCs causing an increase in gene transcription [2, 31. The mechanisms involved in gene repression are less well understood. In some instances, this may be because GR binding to a negative GRE results in steric hindrance to the binding of transcription factors that may induce the same gene. GR may also form complexes with activating transcription factors in the nucleus to inhibit their effect and so modulate genes which contain no GREs. Pro-inflammatory transcription factors, such as AP-1 and NF-KB, can form complexes with activated GR. causing mutual repression of DNA binding or affects interaction with the basal transcription apparatus thereby reducing steroid responsiveness [4-6]. Furthermore, GCs can upregulate the synthesis of I-KB, the cytosolic inhibitor of NF-KB [7]. Conversely, GCs may inhibit the effects of those cytokines that produce their effects on gene transcription via activation of AP-1 or NF-KB. For example, in human lung and in peripheral blood monocytes, TNFa and phorbol esters increase AP-1 and NF-KB binding to DNA and this is inhibited by dexamethasone [6, 81. Cytokines play an important role in chronic inflammation and the pattern of cytokine expression largely determines the nature and persistence of the inflammatory response. GCs have potent inhibitory effects on cytokine mediated inflammation and inhibit the transcription of several cytokines that are relevant in chronic inflammation, including interleukin-1 (IL-l), TNFa, GM-CSF, 11-3, 11-4, IL-5, 11-6 and IL-8 [3]. Other members of the steroid superfamily also act in an analogous manner and, in the case of retinoids, the ligand is able to modulate the ability of the receptor to bind to DNA and to AP-1 [9]. We have, therefore, investigated the ability of two glucocorticoids, dexamethasone and fluticasone, to influence GR-induced inhibition of NF-KB and AP-1 DNA binding and on the release of GM-CSF, an NF-KB-inducible gene in A549 cells. GM-CSF release was measured following stimulation with lng/ml IL-lp. Furthermore, we examined the ability of GR to bind to its DNA binding motif. Lung epithelium-like A549 cells were cultured in Dulbecco’s Modified Eagles medium (DMEM) in the presence of 2% foetal calf serum (FCS) until 70-80% confluent. FCS was removed from the culture medium and cells cultured for a further 2 days. Equal numbers of A549 cells were incubated with a range of concentrations of dexamethasone (Dex) and fluticasone propionate (FP) for 1-24 hours in the presence or absence of lnglml of IL-lp. Control experiments using cells incubated in serum free media alone were run in parallel. At every time point cells were collected by microcentrifugation and nuclear protein extracted [6]. Double stranded oligonucleotides encoding the consensus target sequence for GRE, AP-1 and NF-KB were end-labelled using t2P]-ATP and T4 polynucleotide kinase. l pg of nuclear protein from each sample was incubated with 50,000 cpm of labelled oligonucleotide. Protein-DNA complexes were separated on 6% polyacrylamide gels. The retarded band was detected by autoradiogaphy and quantified by laser densitometry. All band density measurements were then expressed as a percentage of initial binding. Specificity was determined by addition of excess unlabelled double stranded oligonucleotides. GM-CSF concentrations in culture supernatants were measured using a specific GM-CSF ELISA calibrated with human recombinant GM-CSF (0-2000pg/ml). Sensitivity of the assay was 8pglml. FP and Dex both caused a time and dose dependent inhibition of IL1p-stimulated GM-CSF release. Inhibition was first detected at 4 hrs post-steroid treatment and was at maximal levels at all time points from 10-24 hrs. At lOhrs FP caused a dose-dependent inhibition of IL-1pstimulated GM-CSF release with complete inhibition at lO-”M (EC= 5.7il.8 x lo’”), whilst Dex inhibition of IL-1p-stimulated GM-CSF release was found to be maximal at 10aM (EC= 1.2iO.5 x 10.9. This showed a 21 1-fold shift in responsiveness in spite of there being only an Figure 1. The effects of dexamethasone (Dex) and Fluticasone Proprionate (FP) on GMCSF release into the Supernatant of A549 cells.