Fig 1.
Effects of TNF and IL1B on dexamethasone-inducible gene expression in primary human structural lung cells.
A. Primary human bronchial epithelial (HBE) cells were pre-treated for 1 h with 10 ng/ml of tumor necrosis factor-α (TNF) or 1 ng/ml of interleukin 1β (IL1B), before addition of 1 μM dexamethasone (Dex). Cells were harvested at 1, 2, 6 and 18 h after Dex addition. B. Airway smooth muscle (ASM) cells were pre-treated for 1 h with 10 ng/ml of TNF or 1 ng/ml of IL1B, before addition of 1 μM Dex. Cells were harvested at 2 and 6 h after Dex addition. Total RNA was extracted, reverse transcribed to cDNA and RT-PCR performed for: regulator of G-protein signaling 2 (RGS2), TSC22 domain family member 3 (TSC22D3; GILZ), dual specificity phosphatase 1 (DUSP1; MKP1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data (n = 4–5), normalized to GAPDH, are expressed as fold and plotted as means ± S.E. Significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 2.
Effect of TNF on dexamethasone-induced gene expression in BEAS-2B cells.
Human bronchial epithelial, BEAS-2B, cells were pre-treated for 1 h with 10 ng/ml of tumor necrosis factor-α (TNF), before addition of 1 μM dexamethasone (Dex). Cells were harvested 6 h after Dex addition. Total RNA was extracted, reverse transcribed to cDNA and RT-PCR performed for: cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), dual specificity phosphatase 1 (DUSP1; MKP1), regulator of G-protein signaling 2 (RGS2), TSC22 domain family member 3 (TSC22D3; GILZ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data (n = 7), normalized to GAPDH, are expressed as fold and plotted as means ± S.E. Significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. ***, P<0.001.
Fig 3.
Effects of IKK2 and MAPK inhibitors on repression of dexamethasone-induced 2×GRE reporter activation by TNF.
A. BEAS-2B cells stably transfected with a 2×glucocorticoid response element (GRE) luciferase reporter were pre-treated for 30 min with 10 μM of the NF-κB or MAPK pathway inhibitors: PS-1145 (PS; IKBKB), PD098059 (PD; MAP2K1/2), SB203580 (SB; p38 MAPKs) or JNK inhibitor VIII (JNK; JNK MAPKs), before addition of 10 ng/ml of tumor necrosis factor-α (TNF). After 1 h, 10 μM dexamethasone (Dex) was added and cells harvested 6 h later for luciferase assay. Data (n = 7), expressed as a percentage of Dex activation, are plotted as means ± S.E. BEAS-2B 2×GRE cells were pre-treated for 30 min with the indicated concentrations of B. PS or JNK or C. JNK in the presence or absence of 10 μM PS, before addition of 10 ng/ml TNF. After 1 h, 10 μM Dex was added and cells harvested 6 h later for luciferase assay. Data (n = 4–8), expressed as fold activation, are plotted as means ± S.E. Significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. *, P<0.05; **, P<0.01; ***, P<0.001. D+T indicates Dex plus TNF treatment.
Fig 4.
Effects of PKC or PI3K inhibitors on TNF-mediated repression of dexamethasone induced 2×GRE reporter activation.
BEAS-2B 2×GRE cells were pre-treated for 30 min with the indicated concentrations of A. the phosphoinositide 3-kinase (PI3K) inhibitors: LY294002 (LY), PI103 (PI) and Wortmannin (W); or B. the protein kinase C (PKC) inhibitors: Ro 31–8220 (Ro), Gö6976 (Gö) and GF109203X (GF); before addition of 10 ng/ml of tumor necrosis factor-α (TNF). After 1 h, 10 μM dexamethasone (Dex) was added and cells harvested 6 h later for luciferase assay. Data (n = 3–4), expressed as fold activation, are plotted as means ± S.E. D+T indicates Dex plus TNF treatment.
Fig 5.
Effects of TNF on 2×GRE reporter activation induced by NR3C1 ligands.
BEAS-2B 2×GRE cells were pre-treated with 10 ng/ml of TNF for 1 h, before addition of NR3C1 ligands: the glucocorticoids dexamethasone (Dex), fluticasone furoate (FF), fluticasone propionate (FP), budesonide (Bud) and des-ciclesonide (DC); the non-steroidal NR3C1 agonist GSK9027 (GSK) or the selective glucocorticoid receptor agonists (SEGRAs): RU24858 (RU) and GW870086X (GW), at the indicated concentrations. Cells were harvested 6 h after NR3C1 ligand addition for luciferase assay. Data (n = 4–6), expressed as fold activation (Dex) or as a percentage of Dex activation, are plotted as means ± S.E. Statistical significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. At least the top four concentrations of each NR3C1 ligand had statistical significance of P<0.001 (***) relative to the NR3C1 ligand in the presence of TNF.
Table 1.
Effect of NR3C1 ligands on 2×GRE activation in the presence and absence of TNF.
Fig 6.
Effects of TNF and formoterol on 2×GRE activation induced by NR3C1 ligands.
BEAS-2B 2×GRE cells were pre-treated with 10 ng/ml of tumor necrosis factor-α (TNF) for 1 h, before addition of maximally effective concentrations of fluticasone furoate (FF; 100 nM), fluticasone propionate (FP; 100 nM), budesonide (Bud; 100 nM), dexamethasone (Dex; 1 μM), GSK9027 (GSK; 1 μM), RU24858 (RU; 1 μM), des-ciclesonide (DC; 100 nM), GW870086X (GW; 100 nM), in the presence or absence of 10 nM formoterol (Form). Cells were harvested after 6 h for luciferase assays. Data (n = 7), expressed as fold activation, are plotted as means ± S.E. Lines of best fit were added in panel B. Statistical analyses were performed by ANOVA with a Dunnett’s test comparing each NR3C1 ligand alone versus in the presence of TNF and/or Form. *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 7.
Effects of JNK MAPK or IKK2 inhibition on TNF-induced repression of glucocorticoid inducible gene expression.
BEAS-2B cells were pre-treated for 30 min with either 10 or 3 μM of the JNK MAPK inhibitor JNK inhibitor VIII (JNK) or 10 μM of the IKK2 inhibitor PS-1145 (PS), before addition of 10 ng/ml tumor necrosis factor-α (TNF). After 1 h, 1 μM dexamethasone (Dex) and/or 10 nM formoterol (Form) was added and cells were harvested 6 h later. Total RNA was extracted, cDNA synthesized and RT-PCR performed for: cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), dual specificity phosphatase 1 (DUSP1; MKP1), regulator of G-protein signaling 2 (RGS2), TSC22 domain family member 3 (TSC22D3; GILZ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data (n = 9), normalized to GAPDH, are expressed as fold and plotted as means ± S.E. Significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. **, P<0.01. Dexamethasone significantly increased expression of all four genes.
Fig 8.
Effects of TNF and formoterol on gene expression induced by NR3C1 ligands.
BEAS-2B cells were pre-treated for 1 h with 10 ng/ml of tumor necrosis factor-α (TNF), prior to the addition of 10 nM formoterol (Form) and/or the NR3C1 ligands: 1 μM dexamethasone (Dex), 100 nM fluticasone furoate (FF), 100 nM des-ciclesonide (DC) or 100 nM GW870086X (GW). Cells were harvested 6 h after NR3C1 ligand addition. Total RNA was extracted, cDNA synthesized and RT-PCR performed for: cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), dual specificity phosphatase 1 (DUSP1; MKP1), regulator of G-protein signaling 2 (RGS2), TSC22 domain family member 3 (TSC22D3; GILZ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data (n = 5), normalized to GAPDH, are expressed as fold and plotted as means ± S.E. Statistical significance was tested using repeated measures, one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. *, P<0.05; **, P<0.01; ***, P<0.001. Each NR3C1 ligand significantly increased expression of all four genes.