Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms

<div><p>Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the <em>de novo</em> synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent repression by dexamethasone. In conclusion, our data indicate roles for both transrepression and transactivation in the glucocorticoid-dependent repression of inflammatory gene expression. However, transactivation appears to account for the more potent and efficacious mechanism of repression by glucocorticoids on these IL-1β-induced genes.</p> </div

Effect of ORG34517 and GR-specific siRNA on repression of inflammatory gene expression by dexamethasone. A.

<p>A549 cells stably transfected with a 2×GRE reporter were incubated with the indicated concentrations of ORG34517 for 30 min prior to stimulation with increasing concentrations of dexamethasone (Dex) as indicated for 6 h. Cells were then harvested for luciferase assay. Data (n = 4–5) are expressed as a percentage of 1 µM dexamethasone and plotted as means ± SE. <b>B.</b> A549 cells were incubated with either lamin- (control) or GR-specific siRNA for 24 h prior to harvesting for western blot analysis of lamin A/C, GR and GAPDH. Following densitometric analysis, data (n = 5), normalised to GAPDH and expressed as percentage of NS are plotted as means ± SE. Significance, relative to lamin siRNA treated cells, using ANOVA with a Dunnet’s post-test, is indicated: ns, not significant; ***, <i>P</i><0.001. Representative blots are shown. <b>C.</b> A549 cells stably transfected with a 2×GRE reporter were incubated with lamin- (control) or GR-specific siRNA for 24 h prior to stimulation with increasing concentrations of dexamethasone (Dex) as indicated. After 6 h, cells were harvested for luciferase assay. Data (n = 4–5) are expressed as percentage of 1 µM dexamethasone and plotted as means ± SE. <b>D.</b> A549 cells were either not treated or incubated with either ORG34517 for 30 min (left panel of each graph), or lamin- (control) or GR-specific siRNA for 24 h (right panel of each graph) prior to being either not stimulated (data not shown), or treated with IL-1β (1 ng/ml) in the absence (data not shown) or presence of either 0.1 or 1 µM dexamethasone (Dex) for 6 h. Cells were then harvested for real-time PCR analysis of the indicated genes and GAPDH. Data (n = 5–6) normalised to GAPDH and expressed as percentage of IL-1β are plotted as means ± SE. Left panel of each graph: significance relative to IL-1β+Dex was tested using a paired t-test and is indicated: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Right panel of each graph: significance relative to IL-1β+Dex+Lamin siRNA was tested using ANOVA with a Dunnett’s post test (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone.0053936.s009" target="_blank">Table S4</a>) and is indicated: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Effect of IκBαΔN on inflammatory gene expression.

<p><b>A.</b> A549 cells were infected with the NF-κB-dependent reporter, Ad5-NF-κB-luc, in the presence of Ad5-IκBαΔN (0.1–100 MOI) or Ad5-null. After 36 h, cells were stimulated with IL-1β (1 ng/ml) for 6 h before harvesting for luciferase assay and western blot analysis of IκBα and GAPDH. Data (n = 2) expressed as percentage of IL-1β are plotted as mean ± SE. Representative blots are shown. <b>B.</b> A549 cells were infected with 100 MOI of Ad5-IκBαΔN or Ad5-null for 24 h prior to stimulation with IL-1β for 6 h. Cells were harvested for western blot analysis of IκBα and GAPDH. Blots representative of 4 experiments are shown. <b>C.</b> Cells from B were harvested for RNA and SYBR green real-time PCR carried out for GAPDH and the indicated genes. Data (n = 4) normalised to GAPDH and expressed as percentage of IL-1β are plotted as mean ± S.E. Significance relative to IL-1β was tested using AVOVA with a Dunnett’s post-test. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. Genes not significantly repressed by dexamethasone (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g002" target="_blank">Figure 2A</a>) are highlighted in bold font. <b>D.</b> A549 cells infected with the NF-κB-dependent reporter, Ad5-NF-κB-luc, for 36 h were incubated with various concentrations of dexamethasone, as indicated, for 1 h prior to stimulation with IL-1β. After 6 h cells were harvested for luciferase assay. Data (n = 5) expressed as percentage of IL-1β are plotted as mean ± SE.</p

Effect of dexamethasone on inflammatory gene expression.

<p><b>A.</b> The effect of dexamethasone (1 µM) is shown on the induction of inflammatory genes by IL-1β (1 ng/ml) at 6 h. Data from Figures S2 and S3 were combined and the effect of dexamethasone expressed as a percentage of IL-1β for each gene. 100% indicates no effect of dexamethasone. Data (n = 9) are plotted as means ± SE. Genes are listed by descending efficacy to repression by dexamethasone. Statistical analysis was performed by non-parametric paired t-test. * <i>P</i><0.05; ** <i>P</i><0.01. <b>B.</b> Relationship between induction by IL-1β and repression by dexamethasone. The effect of dexamethasone (1 µM) expressed as a percentage of IL-1β is plotted against the fold induction of each gene. Data are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g002" target="_blank">Figure 2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone.0053936.s002" target="_blank">Figure S2</a>. Linear regression was performed using GraphPad Prizm software. <b>C.</b> Repression by dexamethasone is shown for the most highly sensitive gene (IL-1β), two genes showing intermediate sensitivity (EFNA1 & IFIT3 isoform 2) and the lowest sensitivity gene (CFB) (that showed significant repression by 1 µM dexamethasone). Actual EC₅₀ values are indicated. <b>D.</b> Relationship between the effect of 1 µM dexamethasone (efficacy) and the sensitivity (log EC₅₀) of the repression by dexamethasone for individual genes. The effect of dexamethasone (1 µM) expressed as percentage of IL-1β is plotted against the log EC₅₀ for the repression of each gene. Data are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g002" target="_blank">Figure 2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone.0053936.s003" target="_blank">Figure S3</a> respectively. Linear regression was performed using GraphPad Prizm software.</p

Effect of dexamethasone treatment alone on inflammatory mRNA expression.

<p>A549 cells were either not treated or stimulated with IL-1β (1 ng/ml), dexamethasone (Dex) (1 µM) or a combination of the two for 1, 2, 6 or 18 h prior to harvesting for real-time PCR analysis of 39 inflammatory genes and GAPDH. Data (n = 3) normalised to GAPDH are presented as means ± SE for genes showing greater than 2-fold induction by dexamethasone at any time point. Significance relative to non-stimulated samples was tested at time points showing greater than 2-fold induction using a paired t-test and is indicated:</p>*<p>, <i>P</i><0.05;</p>**<p>, <i>P</i><0.01;</p>***<p>, <i>P</i><0.001.</p

Effect of CHX on dexamethasone-dependent repression of inflammatory gene expression.

<p>A549 cells were treated with IL-1β (1 ng/ml) (not shown), or IL-1β and dexamethasone (dex) (1 µM) in the absence or presence of cycloheximide (CHX) (100 µg/ml) as indicated, for 4 h. Cells were then harvested for RNA and real-time PCR carried out for the indicated genes and GAPDH. Data (n = 4) normalised to GAPDH and plotted as percentage of IL-1β or IL-1β+CHX are expressed as mean ± S.E. Significance, relative to IL-1β+dex, using a paired t-test is indicated; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. <b>A.</b> Full reversal (significantly different from IL-1β+dex and from IL-1β+CHX), <b>B.</b> partial reversal (significantly different from only IL-1β+dex) and <b>C.</b> no reversal (not significantly different from IL-1β+dex) of dexamethasone-dependent repression by CHX.</p

Effect of IL-1β and dexamethasone on inflammatory gene expression. A.

<p>A549 cells were either not stimulated (NS) or treated with IL-1β (1 ng/ml), dexamethasone (dex) (1 µM) or a combination of the two for 1, 2, 6 and 18 h. Cells were then harvested for RNA and real-time PCR was carried out for the indicated genes and GAPDH. Data (n = 3), normalised to GAPDH are expressed as either log₂ fold over NS at 1 h (left panel) or as percentage of IL-1β (right hand panel) and plotted as means. Genes are grouped based on expression patterns: ‘Early-phase’ genes are those which have a peak of expression at 1 or 2 h (top two graphs); ‘Late-phase’ genes have a peak of expression at 6 h or later with less than 50% of that peak expression observed at 1 or 2 h (bottom two graphs); and ‘Intermediate’ genes are those that fall into neither of the above categories (middle two graphs). <b>B.</b> Effect of dexamethasone from right hand panel of A is plotted at each time point for all genes repressed by dexamethasone. Significance was tested using one-way ANOVA with a Bonferroni post-test and is indicated: *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

Relationship between the sensitivity and potency of repression by dexamethasone and the reversal by cycloheximide in A549 cells. A.

<p>Data showing the effect of 1 µM dexamethasone (as % IL-1β) plotted against the EC₅₀ for repression of each target mRNA by dexamethasone (i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g002" target="_blank">Figure 2D</a>), were overlaid with the effect of cycloheximide (CHX) on that repression (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g005" target="_blank">Figure 5</a>). The logEC₅₀ values for repression by dexamethasone of mRNAs showing: <b>B,</b> full reversal, or; <b>C,</b> full+partial reversal of this repression by cycloheximide were compared with the logEC₅₀ values for mRNAs showing no reversal of dexamethasone-dependent repression by cycloheximide. Likewise, the effect of dexamethasone at 1 µM, expressed as a percentage of IL-1β, was compared for mRNAs showing: <b>D,</b> full reversal, or; <b>E,</b> full+partial reversal of this repression by cycloheximide were compared with mRNAs showing no reversal of dexamethasone-dependent repression by cycloheximide. Statistical analyses were performed by unpaired t-test. * <i>P</i><0.05, *** <i>P</i><0.001. <b>F.</b> Data showing the effect of 1 µM dexamethasone at 1, 2, 6 and 18 h (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053936#pone-0053936-g001" target="_blank">Figure 1</a>) are plotted for the group of genes showing reversal of repression by cycloheximide (left graph) and the group of genes showing no reversal (right graph). Statistical analysis was performed using ANOVA with a Bonferroni post-test and is indicated: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Quantitative Proteomics of Bronchoalveolar Lavage Fluid in Idiopathic Pulmonary Fibrosis

The proteomic analysis of bronchoalveolar lavage fluid (BALF) can give insight into pulmonary disease pathology and response to therapy. Here, we describe the first gel-free quantitative analysis of BALF in idiopathic pulmonary fibrosis (IPF), a chronic and fatal scarring lung disease. We utilized two-dimensional reversed-phase liquid chromatography and ion-mobility-assisted data-independent acquisition (HDMSE) for quantitation of >1000 proteins in immunodepleted BALF from the right middle and lower lobes of normal controls and patients with IPF. Among the analytes that were increased in IPF were well-described mediators of pulmonary fibrosis (osteopontin, MMP7, CXCL7, CCL18), eosinophil- and neutrophil-derived proteins, and proteins associated with fibroblast foci. For additional discovery and targeted validation, BALF was also screened by multiple reaction monitoring (MRM), using the JPT Cytokine SpikeMix library of >400 stable isotope-labeled peptides. A refined MRM assay confirmed the robust expression of osteopontin, and demonstrated, for the first time, upregulation of the pro-fibrotic cytokine, CCL24, in BALF in IPF. These results show the utility of BALF proteomics for the molecular profiling of fibrotic lung diseases and the targeted quantitation of soluble markers of IPF. More generally, this study addresses critical quality control measures that should be widely applicable to BALF profiling in pulmonary disease