Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation: preferential targetting of NF-kappaB and lack of I-kappaB involvement

Abstract

1. Glucocorticoids are highly effective in controlling chronic inflammatory diseases, such as asthma and rheumatoid arthritis, but the exact molecular mechanism of their anti-inflammatory action remains uncertain. They act by binding to a cytosolic receptor (GR) resulting in activation or repression of gene expression. This may occur via direct binding of the GR to DNA (transactivation) or by inhibition of the activity of transcription factors such as AP-1 and NF-kappaB (transrepression). 2. The topically active steroids fluticasone propionate (EC50= 1.8 x 10(-11) M) and budesonide (EC50=5.0 x 10(-11) M) were more potent in inhibiting GM-CSF release from A549 cells than tipredane (EC50 = 8.3 x 10(-10)) M), butixicort (EC50 = 3.7 x 10(-8) M) and dexamethasone (EC50 = 2.2 x 10(-9) M). The anti-glucocorticoid RU486 also inhibited GM-CSF release in these cells (IC50= 1.8 x 10(-10) M). 3. The concentration-dependent ability of fluticasone propionate (EC50 = 9.8 x 10(-10) M), budesonide (EC50= 1.1 x 10(-9) M) and dexamethasone (EC50 = 3.6 x 10(-8) M) to induce transcription of the beta2-receptor was found to correlate with GR DNA binding and occurred at 10-100 fold higher concentrations than the inhibition of GM-CSF release. No induction of the endogenous inhibitors of NF-kappaB, IkappaBalpha or I-kappaBbeta, was seen at 24 h and the ability of IL-1beta to degrade and subsequently induce IkappaBalpha was not altered by glucocorticoids. 4. The ability of fluticasone propionate (IC50=0.5 x 10(-11) M), budesonide (IC50=2.7 x 10(-11) M), dexamethasone (IC50=0.5 x 10(-9) M) and RU486 (IC50=2.7 x 10(-11) M) to inhibit a 3 x kappaB was associated with inhibition of GM-CSF release. 5. These data suggest that the anti-inflammatory properties of a range of glucocorticoids relate to their ability to transrepress rather than transactivate genes.

Figure 1

(a) Concentration-dependent inhibition of interleukin (IL)-1β (1 ng ml⁻¹)-stimulated granulocyte-macrophage colony stimulating factor (GM-CSF) release into the media from A549 cells at 24 h following fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex) treatment. (b) Concentration-dependent inhibition of IL-1β (1 ng ml⁻¹)-stimulated GM-CSF release from A549 cells at 24 h following treatment with the anti-glucocorticoid RU486. (c) The effect of low concentration (10⁻⁹ M) RU486 (RU) treatment on the inhibition of IL-1β-stimulated GM-CSF release by 10⁻¹⁰ M FP, Bud and Dex. (d) The effects of increasing concentrations of phenylarsine oxide (PAO) on IL-1β (1 ng ml⁻¹)-stimulated induction of GM-CSF release into culture medium at 24 h. Results are plotted as the means±s.e.means of the percentage of maximal IL-1β-stimulated GM-CSF release in the absence any drug. n=4–7 for each data point except in (c) where results are the mean of two independent experiments.

Figure 1

(a) Concentration-dependent inhibition of interleukin (IL)-1β (1 ng ml⁻¹)-stimulated granulocyte-macrophage colony stimulating factor (GM-CSF) release into the media from A549 cells at 24 h following fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex) treatment. (b) Concentration-dependent inhibition of IL-1β (1 ng ml⁻¹)-stimulated GM-CSF release from A549 cells at 24 h following treatment with the anti-glucocorticoid RU486. (c) The effect of low concentration (10⁻⁹ M) RU486 (RU) treatment on the inhibition of IL-1β-stimulated GM-CSF release by 10⁻¹⁰ M FP, Bud and Dex. (d) The effects of increasing concentrations of phenylarsine oxide (PAO) on IL-1β (1 ng ml⁻¹)-stimulated induction of GM-CSF release into culture medium at 24 h. Results are plotted as the means±s.e.means of the percentage of maximal IL-1β-stimulated GM-CSF release in the absence any drug. n=4–7 for each data point except in (c) where results are the mean of two independent experiments.

Figure 2

(a) Representative electrophoretic mobility shift assay showing the concentration-dependent effect of fluticasone propionate (FP), Budesonide (Bud) and dexamethasone (Dex) on glucocorticoid receptor (GR)-induced activation as represented by increased DNA binding (GRE binding) (arrowed) within the nucleus after 2 h incubation. (b) Supershift assay of dexamethasone (10⁻⁶ M)-stimulated GR DNA binding. Increased DNA binding is seen following dexamethasone treatment (lane 2). Pre-incubation of retarded complexes with an anti-GR antibody (lane 3) shows specific enhanced retardation of GR/GRE band. Specificity of binding was indicated by the addition of 100 fold excess unlabelled oligonucleotide (lane 4). Unbound oligonucleotide is indicated by an arrow at the bottom of the gel. (c) Densitometric analysis of the retarded bands in (a) and corrected for maximal band intensity showing the concentration-dependent increase in GRE binding following 2 h incubation with FP, Bud and Dex within the nucleus as a percentage of the maximal increase observed.

Figure 2

(a) Representative electrophoretic mobility shift assay showing the concentration-dependent effect of fluticasone propionate (FP), Budesonide (Bud) and dexamethasone (Dex) on glucocorticoid receptor (GR)-induced activation as represented by increased DNA binding (GRE binding) (arrowed) within the nucleus after 2 h incubation. (b) Supershift assay of dexamethasone (10⁻⁶ M)-stimulated GR DNA binding. Increased DNA binding is seen following dexamethasone treatment (lane 2). Pre-incubation of retarded complexes with an anti-GR antibody (lane 3) shows specific enhanced retardation of GR/GRE band. Specificity of binding was indicated by the addition of 100 fold excess unlabelled oligonucleotide (lane 4). Unbound oligonucleotide is indicated by an arrow at the bottom of the gel. (c) Densitometric analysis of the retarded bands in (a) and corrected for maximal band intensity showing the concentration-dependent increase in GRE binding following 2 h incubation with FP, Bud and Dex within the nucleus as a percentage of the maximal increase observed.

Figure 2

(a) Representative electrophoretic mobility shift assay showing the concentration-dependent effect of fluticasone propionate (FP), Budesonide (Bud) and dexamethasone (Dex) on glucocorticoid receptor (GR)-induced activation as represented by increased DNA binding (GRE binding) (arrowed) within the nucleus after 2 h incubation. (b) Supershift assay of dexamethasone (10⁻⁶ M)-stimulated GR DNA binding. Increased DNA binding is seen following dexamethasone treatment (lane 2). Pre-incubation of retarded complexes with an anti-GR antibody (lane 3) shows specific enhanced retardation of GR/GRE band. Specificity of binding was indicated by the addition of 100 fold excess unlabelled oligonucleotide (lane 4). Unbound oligonucleotide is indicated by an arrow at the bottom of the gel. (c) Densitometric analysis of the retarded bands in (a) and corrected for maximal band intensity showing the concentration-dependent increase in GRE binding following 2 h incubation with FP, Bud and Dex within the nucleus as a percentage of the maximal increase observed.

Figure 3

(a) Western blot analysis of β₂-receptor (β₂R) expression at 24 h following increasing concentrations of fluticasone propionate (FP), budesonide (Bud), dexamethasone (dex) or RU486. The single 47 kD band representing the β₂-receptor is indicated by the arrow. Incubation with control media does not affect β₂-receptor expression. (b) Graphical representation of the results shown in (a). Results are shown as the percentage change in β₂-receptor band density compared to control untreated cells and are representative of four individual experiments and are reported as the means±s.e.means.

Figure 3

(a) Western blot analysis of β₂-receptor (β₂R) expression at 24 h following increasing concentrations of fluticasone propionate (FP), budesonide (Bud), dexamethasone (dex) or RU486. The single 47 kD band representing the β₂-receptor is indicated by the arrow. Incubation with control media does not affect β₂-receptor expression. (b) Graphical representation of the results shown in (a). Results are shown as the percentage change in β₂-receptor band density compared to control untreated cells and are representative of four individual experiments and are reported as the means±s.e.means.

Figure 4

Western blot analysis of the time course of I-κBα expression following 24 h treatment with various concentrations of fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex). Concentrations are reported as −log of Molar concentrations. (b) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹) in the presence and absence of the proteosome inhibitor CBZ-leucine-leucine-leucinal (LLLal) (50 μM) I-κBα is indicated by the arrow. (c) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹). The effects of fluticasone propionate (FP, 10⁻¹⁰ M), budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBα phosphorylation, degradation and subsequent induction are shown. I-κBα and the slower migrating phosphorylated form of I-κBα are indicated by the arrows. (d) The lack of effect of fluticasone propionate (FP, 10⁻¹⁰ M) budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBβ phosphorylation, degradation and subsequent induction over 0–8 h are shown. Results are representative of four individual experiments.

Figure 4

Western blot analysis of the time course of I-κBα expression following 24 h treatment with various concentrations of fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex). Concentrations are reported as −log of Molar concentrations. (b) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹) in the presence and absence of the proteosome inhibitor CBZ-leucine-leucine-leucinal (LLLal) (50 μM) I-κBα is indicated by the arrow. (c) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹). The effects of fluticasone propionate (FP, 10⁻¹⁰ M), budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBα phosphorylation, degradation and subsequent induction are shown. I-κBα and the slower migrating phosphorylated form of I-κBα are indicated by the arrows. (d) The lack of effect of fluticasone propionate (FP, 10⁻¹⁰ M) budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBβ phosphorylation, degradation and subsequent induction over 0–8 h are shown. Results are representative of four individual experiments.

Figure 4

Western blot analysis of the time course of I-κBα expression following 24 h treatment with various concentrations of fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex). Concentrations are reported as −log of Molar concentrations. (b) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹) in the presence and absence of the proteosome inhibitor CBZ-leucine-leucine-leucinal (LLLal) (50 μM) I-κBα is indicated by the arrow. (c) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹). The effects of fluticasone propionate (FP, 10⁻¹⁰ M), budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBα phosphorylation, degradation and subsequent induction are shown. I-κBα and the slower migrating phosphorylated form of I-κBα are indicated by the arrows. (d) The lack of effect of fluticasone propionate (FP, 10⁻¹⁰ M) budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBβ phosphorylation, degradation and subsequent induction over 0–8 h are shown. Results are representative of four individual experiments.

Figure 4

Western blot analysis of the time course of I-κBα expression following 24 h treatment with various concentrations of fluticasone propionate (FP), budesonide (Bud) and dexamethasone (Dex). Concentrations are reported as −log of Molar concentrations. (b) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹) in the presence and absence of the proteosome inhibitor CBZ-leucine-leucine-leucinal (LLLal) (50 μM) I-κBα is indicated by the arrow. (c) Western blot analysis of the time course of I-κBα expression following up to 90 min treatment with IL-1β (1 ng ml⁻¹). The effects of fluticasone propionate (FP, 10⁻¹⁰ M), budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBα phosphorylation, degradation and subsequent induction are shown. I-κBα and the slower migrating phosphorylated form of I-κBα are indicated by the arrows. (d) The lack of effect of fluticasone propionate (FP, 10⁻¹⁰ M) budesonide (Bud, 10⁻¹⁰ M) and dexamethasone (Dex, 10⁻⁹ M) on IL-1β-stimulated I-κBβ phosphorylation, degradation and subsequent induction over 0–8 h are shown. Results are representative of four individual experiments.

Figure 5

Western blot analysis of immunoprecipitated p65 and GR complexes. Cells were treated for 30 min with a combination of IL-1β (1 ng ml⁻¹) and dexamethasone (10⁻⁶ M) before cell were lysis. Total cell extracts were immunoprecipitated with an anti-human p65 antibody (lane 1), an anti-human GR antibody (lane 4) or with pre-immune serum (lanes 2 and 3) before separation by 10% PAGE and detection of bands by either anti-human GR antibody (lanes 1 and 2) or anti-human p65 antibody (lanes 3 and 4). The specific GR or p65 bands are indicated arrows. The 50 kDa IgG heavy chain is detected in all samples and is also arrowed. Molecular weight markers are as indicated. The results are representative of three independent experiments.

Figure 6

(a) Inhibition of an IL-1β (1 ng ml⁻¹)-stimulated 3×κB-driven luciferase reporter gene by fluticasone propionate (FP), budesonide (Bud), dexamethasone (Dex) and RU486. Results are expressed as relative light units/unit β-galactsidase activity (means±s.e.mean) and represent the results of at least four independent experiments. (b) Inhibition of IL-1β (1 ng ml⁻¹)-stimulated 6×TRE-Luc reporter gene by fluticasone propionate (FP) and dexamethasone (Dex). Results are expressed as relative light units/unit β-galactsidase activity (means±s.e.mean) and represent the results of at least four independent experiments.