The long-acting β2 -adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner

Abstract

Background and purpose: Inhaled glucocorticoid (ICS)/long-acting β2 -adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism.

Experimental approach: BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes.

Key results: Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest.

Conclusions and implications: These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues.

Figure 1

Effect of indacaterol on GRE-dependent transcription in 2× GRE BEAS-2B reporter cells. Cells were treated with FF (A), Dex (B), DC (C), GW (D), Mif (E) or Org (F) at the concentrations indicated in the absence and presence of indacaterol (100 nM), which was added concurrently. In panels G–L, cells were treated with indacaterol over the concentration ranges indicated in the presence of a maximally effective concentration of each glucocorticoid that was determined from the data shown in panels A–F. At 6 h cells were harvested, luciferase activity was determined and E/[A] curves were constructed. Data points represent the mean ± SEM of n independent determinations. The fold values in each panel indicate the maximal fold enhancement of GRE-dependent transcription produced by indacaterol (100 nM). The dashed lines in panels G–L indicate luciferase activity produced by glucocorticoid alone.

Figure 2

Effect of indacaterol on the intrinsic activity values of a panel of GR ligands in promoting GRE-dependent transcription in 2× GRE BEAS-2B reporter cells. Cells were treated with GR ligands at the concentrations indicated in the absence (panel A) and presence (panel B) of indacaterol (100 nM) and E/[A] curves were then constructed. Panel C shows the relationship between the intrinsic activity values of each GR ligand in the absence and presence of indacaterol, with FF being assigned a value of 1. The dashed line indicates line of identity. Data points represent the mean ± SEM of four to seven independent determinations (see Table 2b).

Figure 3

Effect of indacaterol on GSK 9027-induced, GRE-dependent transcription in BEAS-2B reporter cells. In panel A, cells were treated with GSK 9027 at the concentrations indicated in the absence and presence of indacaterol (100 nM), which was added concurrently. Dex (1 μM) and FF (100 nM) in the absence and presence of indacaterol were included as comparators. In panel B, cells were treated with indacaterol (3 pM to 100 nM) in the presence of a maximally effective concentration of GSK 9027 (3 μM) determined from the data shown in panel A. At 6 h cells were harvested, luciferase activity was determined and E/[A] curves were constructed. Data points represent the mean ± SEM of n independent determinations. The fold values in each panel indicate the maximal fold enhancement of GRE-dependent transcription produced by indacaterol (100 nM). The dashed line in panel B indicates luciferase activity produced by GSK 9027 alone.

Figure 4

Influence of agonist intrinsic activity on the ability of indacaterol to enhance GRE-dependent transcription in 2× GRE BEAS-2B reporter cells. Panel A shows the relationship between GRE-dependent luciferase activity produced by seven GR ligands in the absence and presence of indacaterol (100 nM), which was linear. Panel B shows that the fold enhancement by indacaterol of the maximal transcription produced by each GR ligand as a saturable function of agonist intrinsic activity.

Figure 5

Analysis of Dex and FF E/[A] curve data in the absence and presence of Dex-Mes by operational model fitting. 2× GRE BEAS-2B reporter cells were treated with Dex-Mes (10 nM or 30 min) or vehicle. The cells were washed in Dex-Mes-free medium and E/[A] curves constructed to Dex (panels A and C) or FF (panels B and D) in the absence and presence of indacaterol (100 nM) as described in the legend to Figure 1. The model parameter estimates for Dex and FF are provided in Table 4. The bars in each panel show the effect of Dex-Mes, indacaterol, Dex-Mes/indacaterol, GR agonist and GR agonist after treatment of cells with Dex-Mes respectively. The downward arrows in each panel reflect the percentage inhibition of luciferase activity produced by Dex-Mes. The system maximum parameter, E_m, and upper asymptote of control E/[A] curves are shown in each panel. Data points represent the mean ± SEM of n independent determinations.

Figure 6

Comparative effects of indacaterol on gene expression produced by a panel of GR ligands. 2× GRE BEAS-2B reporter cells were treated with FF (100 nM), Dex (1 μM), GSK 9027 (3 μM), DC (100 nM), GW (1 μM), Mif (1 μM) or Org (1 μM) in the absence and presence of indacaterol (100 nM). At 6 h, total RNA was extracted, reverse transcribed and the resulting cDNA subjected to real-time PCR using primer pairs specific for PDK4, p57^kip2, CRISPLD2 and GILZ. Data are the mean ± SEM of n independent determinations and are expressed a ratio to GAPDH. *P < 0.05, significant enhancement of gene expression produced by indacaterol; **P < 0.05, significant induction of gene expression relative to untreated (NS) or indacaterol-treated cells. Data were analysed by repeated measures, one-way anova/Tukey's multiple comparisons test. ^aP < 0.05, significant difference in gene expression relative to that produced by fluticasone furoate.

Figure 7

Effect of GR ligand intrinsic activity on the ability of indacaterol to enhance the expression of PDK4 and p57^kip2. Panels A and B show the relationship between the expression of PDK4 and p57^kip2 produced by seven GR agonists in the absence and presence of indacaterol (100 nM), which was linear. Panels C and D show that the fold enhancement by indacaterol of the maximal transcription produced by each GR agonist produced by indacaterol was a saturable function of agonist intrinsic activity.

Figure 8

Effect of indacaterol on the ability of FF and GW to promote gene expression. 2× GRE BEAS-2B reporter cells were treated with FF (panels A–D) and GW (panels E–H) in the absence and presence of a maximally effective concentration of indacaterol (100 nM). At 6 h, total RNA was extracted, reverse transcribed and the resulting cDNA subjected to real-time PCR using primer pairs specific for GILZ, CRISPLD2, p57^kip2 and PDK4. Data are expressed as a ratio to GAPDH and presented as E/[A] curves. Each bar and data point represents the mean ± SEM of n independent determinations. The dashed line in each panel defines gene expression produced by indacaterol alone. Quantification and statistical analyses of these data are presented in Table 6. The upward arrows in each panel show the fold enhancement by indacaterol of gene expression produced by a maximally effective concentration of GR agonist.

Figure 9

Effect of indacaterol on the expression of p57^kip2 produced by FF. The graph in Figure 8C has been redrawn to illustrate that the addition of indacaterol (100 nM) to FF (10 pM to 1 μM) enhanced the expression of a representative glucocorticoid-inducible gene, p57^kip2, in a ‘steroid-sparing’ manner. In these cells, indacaterol had negligible activity on the expression of p57^kip2 (black bar) but markedly augmented the effect of FF from 11.5- to 31.5-fold at the p[A]₉₅ (a 270% increase; pink line). Indacaterol was also steroid sparing in these cells. Thus, in the presence of indacaterol, an 11.5-fold increase in p57^kip2 mRNA levels was produced at a concentration of FF that was 10-fold lower (green line). Ind, indacaterol.

Figure 10

Hypothetical Venn diagram illustrating a mechanism that may contribute to the clinical superiority of ICS/LABA combination therapy. Two ‘sets’ of genes are shown that are induced by glucocorticoid (A) or LABA (B). Some of these genes are only induced by one component of the combination therapy, while others are induced by both glucocorticoid and LABA. According to the model, A ∩ B is suggested to be primarily responsible for the enhanced effect of ICS/LABA combination therapy through the ability of both components to interact in an additive or positive cooperative manner. However, disease-modifying genes that are induced by ICS alone (e.g. GILZ) and LABA alone (C5AR1) may also play an important role such that it is the ‘union’ of both gene populations (A U B) that delivers the clinical effect. The Venn diagram is populated with examples of genes that fall within set A, set B or A ∩ B. The size of each set and the area of intersection that defines the union are unknown. It is important to note that potential adverse effect genes (e.g. PDK4, not shown) will also constitute part of the Venn diagram and could, by the same mechanism, increase side effects. Similarly, there may be populations of LABA-inducible, anti-inflammatory genes that are inhibited by ICS. Currently, in vivo studies in humans to determine if a LABA can augment glucocorticoid-induced gene expression in the airways have not been performed. C5AR1, complement component 5 receptor 1 (Köhl et al., 2006); CD200, cluster of differentiation 200 (Snelgrove et al., 2008); CRISPLD2, cysteine-rich secretory protein LCCL (limulus clotting factor C, cochlin, lgl1) domain-containing 2 (Vasarhelyi et al., , Wang et al., ; Himes et al., 2014); GILZ, glucocorticoid leucine zipper (Eddleston et al., ; Ayroldi and Riccardi, 2009); DUSP-1, MAPK phosphatase-1 (Korhonen and Moilanen, 2014); p57^kip2, kinase inhibitor protein 2 of 57 kDa (Samuelsson et al., 1999); RGS2, regulator of G-protein signalling 2 (Holden et al., 2011; 2014).