Molecular mechanisms of anti-inflammatory glucocorticoid action: Evidence for a role of glucocorticoid-inducible genes and implications for glucocorticoid insensitivity

The anti-inflammatory activity of glucocorticoids, in diseases such as asthma, is attributed to their ability to reduce the expression of multiple inflammatory mediators. While glucocorticoid receptor (NR3C1)-mediated transcriptional activation, or transactivation, was believed to primarily mediate side-effects, accumulating evidence indicates that transactivation is also important for the inhibition of inflammatory gene expression. This illustrates the need to investigate possible functional roles for specific glucocorticoid-inducible genes. In this thesis, a repressive role for DUSP1, a phosphatase that inhibits MAPKs, was investigated. DUSP1 was induced by IL1B and dexamethasone in both human pulmonary A549 and primary HBE cells. IL1B-induced DUSP1 negatively regulated MAPK activity and in the further presence of dexamethasone, DUSP1 played a transient, typically partial, role in repressing expression of inflammatory genes, including CXCL1, CXCL2 and PTGS2. Thus, additional glucocorticoid-induced gene products are necessary for repression. Regulation of the mRNA destabilizing protein, ZFP36, by DUSP1 was examined. Following the loss of DUSP1, ZFP36 expression was enhanced and this attenuated IL1B-induced TNF expression. Despite a modest ability of dexamethasone to induce ZFP36, thereby off-setting loss of ZFP36 due to reduced MAPK activity, neither silencing of dexamethasone-induced ZFP36, DUSP1, nor both together, prevented repression of TNF by dexamethasone. Finally, while DUSP1 over-expression attenuated IL1B-induced expression of many inflammatory genes (e.g. IL8, CSF2 etc.), others, including, the inflammatory transcription factor, IRF1, and downstream target genes, such as CXCL10, were profoundly enhanced. While MAPK inhibition prolonged IRF1 expression, silencing of IL1B plus dexamethasone-induced DUSP1 reduced IRF1 and CXCL10 expression. Since, CXCL10 expression was largely unaffected by dexamethasone, these data suggest a mechanism whereby dexamethasone-induced DUSP1 expression maintains CXCL10 expression. In conclusion, this study demonstrates interlinked counterregulatory networks, in which IL1B-induced DUSP1 and ZFP36 regulate MAPK activation and inflammatory gene expression respectively. The transient and partial repressive effect of dexamethasone-induced DUSP1 on only few IL1B-induced inflammatory genes provides a rational for functional screening of glucocorticoid-inducible genes that may show repressive functions. Furthermore, by switching off MAPKs, DUSP1 maintains IRF1 expression and may contribute to glucocorticoid insensitivity

TNF Mediates the Sustained Activation of Nrf2 in Human Monocytes

Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-a. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-?B. In this study, we investigated the regulation of NF-E2–related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-?B activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses

Cytokine-Induced Loss of Glucocorticoid Function: Effect of Kinase Inhibitors, Long-Acting β2-Adrenoceptor Agonist and Glucocorticoid Receptor Ligands

<div><p>Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2×glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally, formoterol-enhanced 2×GRE reporter activity was also proportional to agonist efficacy and functionally reversed repression by TNF. As similar effects were apparent on glucocorticoid-induced gene expression, the most effective strategy to overcome glucocorticoid resistance in this model was addition of formoterol to high efficacy NR3C1 agonists.</p></div

Effects of TNF and IL1B on dexamethasone-inducible gene expression in primary human structural lung cells.

<p>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. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Effect of TNF on dexamethasone-induced gene expression in BEAS-2B cells.

<p>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. ***, <i>P</i><0.001.</p

Effects of IKK2 and MAPK inhibitors on repression of dexamethasone-induced 2×GRE reporter activation by TNF.

<p>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. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. D+T indicates Dex plus TNF treatment.</p

Effects of JNK MAPK or IKK2 inhibition on TNF-induced repression of glucocorticoid inducible gene expression.

<p>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. **, <i>P</i><0.01. Dexamethasone significantly increased expression of all four genes.</p

Effects of TNF and formoterol on 2×GRE activation induced by NR3C1 ligands.

<p>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. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Effects of TNF and formoterol on gene expression induced by NR3C1 ligands.

<p>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. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Each NR3C1 ligand significantly increased expression of all four genes.</p