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. 2015 Sep 15;5:14086.
doi: 10.1038/srep14086.

The Glucocorticoid Mometasone Furoate Is a Novel FXR Ligand That Decreases Inflammatory but Not Metabolic Gene Expression

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Free PMC article

The Glucocorticoid Mometasone Furoate Is a Novel FXR Ligand That Decreases Inflammatory but Not Metabolic Gene Expression

Ingrid T G W Bijsmans et al. Sci Rep. .
Free PMC article

Abstract

The Farnesoid X receptor (FXR) regulates bile salt, glucose and cholesterol homeostasis by binding to DNA response elements, thereby activating gene expression (direct transactivation). FXR also inhibits the immune response via tethering to NF-κB (tethering transrepression). FXR activation therefore has therapeutic potential for liver and intestinal inflammatory diseases. We aim to identify and develop gene-selective FXR modulators, which repress inflammation, but do not interfere with its metabolic capacity. In a high-throughput reporter-based screen, mometasone furoate (MF) was identified as a compound that reduced NF-κB reporter activity in an FXR-dependent manner. MF reduced mRNA expression of pro-inflammatory cytokines, and induction of direct FXR target genes in HepG2-GFP-FXR cells and intestinal organoids was minor. Computational studies disclosed three putative binding modes of the compound within the ligand binding domain of the receptor. Interestingly, mutation of W469A residue within the FXR ligand binding domain abrogated the decrease in NF-κB activity. Finally, we show that MF-bound FXR inhibits NF-κB subunit p65 recruitment to the DNA of pro-inflammatory genes CXCL2 and IL8. Although MF is not suitable as selective anti-inflammatory FXR ligand due to nanomolar affinity for the glucocorticoid receptor, we show that separation between metabolic and anti-inflammatory functions of FXR can be achieved.

Figures

Figure 1
Figure 1. Luciferase reporter assay identifies 5 compounds decreasing TNFα-induced NF-κB transcriptional activity.
Schematic representation of the molecular FXR actions in regulation of bile salt, glucose and fat metabolism via direct DNA binding (A), and ameliorating inflammation via tethering transrepression of NF-κB (B). Ligand (green triangle) activated FXR binds to FXR responsive elements (FXREs), thereby activating target genes involved in bile salt homeostasis (SHP, IBABP), glucose (PEPCK, G6Pase), and fat metabolism (SREBP1C, FAS). Binding of NF-κB to its responsive element (NF-κB RE) results in expression of pro-inflammatory cytokines, such as IL8 and MCP-1. FXR binding to NF-κB inhibits this activity, thereby decreasing pro-inflammatory cytokine expression. We have set up an automated high-throughput NF-κB luciferase reporter assay to test FXR-dependent reduction of NF-κB activity (C). We screened the Prestwick library containing 1,200 FDA approved drugs (yellow triangle) using this assay (left panel). Candidate drugs were subsequently tested for IBABP and SHP transactivation. Figures D-G depict hit selection. Figures D and E show the overall view of the screen. Indicated with black dots are the 34 drugs reducing TNFα-induced NF-κB transcriptional activity significantly (p < 0.05) (E). Low renilla values were considered to reflect poor transfection efficiency or cytotoxicity and were therefore eliminated, leaving 5 compounds significantly reducing NF-κB activity (indicated with black dots surrounded by orange circles. D: DMSO; G+T: GW4064 + TNFα; G: GW4064; T: TNFα (E). Figure F depicts the chemical structures of the five candidate compounds. Flowchart of hit selection is shown in (G).
Figure 2
Figure 2. MF is an anti-inflammatory FXR modulator.
(A) Validation of NF-κB luciferase reporter assay. HEK293T cells transfected with NF-κB reporter, expression plasmids for FXR and RXR, and pTK-renilla construct were treated with DMSO, GW4064 (1 μM), TNFα (5 ng/ml), GW4064 plus TNFα, or the indicated compounds (10 μM) in the presence of TNFα, for 24 hours. Cilnidipine (C), mometasone furoate (MF), nicardipine hydrochloride (NH), quinacrine dihydrochloride dehydrate (QDD), and topotecan (T) significantly reduced TNFα-induced NF-κB activity. The reporter assay was performed in quadruplicate in three independent experiments. Each bar represents mean ± SD of one representative experiment. *p < 0.001 as compared to TNFα treated cells. (B) FXR-dependent reduction of NF-κB transcriptional activity. The assay in (A) was repeated with empty vector (EV; white bars) and FXR overexpressing cells (black bars). Data are normalized to the EV activity for each compound. (C) Dose-response curve. MF treatment reduced TNFα-induced NF-κB activity in a dose-dependent manner, with an IC50 value of 1.4 μM. IC50 values of CDCA and GW4064 are 7.9 μM and 3.5 nM respectively. (D) Transactivation reporter assay SHP (left panel) and IBABP promoters (right panel). HEK293T cells transfected with SHP or IBABP promoter constructs, FXR and RXR, and renilla, were treated with DMSO, 1 μM GW4064, or 10 μM MF for 24 hours. Data presented show one representative experiment of 4 performed experiments. Each bar represents mean ± SD. *p < 0.001 compared to GW4064 treated cells. (E) FXR coactivator recruitment assay (AlphaScreen). Ligand binding domain of FXR (FXR-LBD) was incubated with increasing amounts of MF or CDCA to examine SRC-1 recruitment. Assay performed in triplicate. One representative experiment is shown.
Figure 3
Figure 3. MF reduces pro-inflammatory gene expression in HepG2 cells and intestinal organoids.
Endogenous FXR target gene expression in HepG2 cells stably overexpressing GFP (HepG2-GFP; white bars) or GFP-FXR (HepG2-GFP-FXR; black bars). (A) Cells were treated in triplicate with DMSO, 1 μM GW4064, 10 μM MF, 5 ng/ml TNFα, GW4064 plus TNFα, or TNFα plus MF for 24 hours. IL8, MCP-1, and CXCL2 mRNA expression was analyzed by qRT-PCR in duplicate. (B) HepG2-GFP and HepG2-GFP-FXR cells were treated with DMSO, 1 μM GW4064 or 10 μM MF in triplicate for 24 hours. SHP, FGF19, KNG1, SDC1 and ICAM1 mRNA expression was analyzed by qRT-PCR in duplicate. Each bar represents mean ± SD. (C,D) Small intestine derived organoids from 3WT and FXR−/− mice were treated with DMSO, 1 μM GW4064, 10 μM MF, 5 ng/ml TNFα, GW4064 plus TNFα, or TNFα plus MF for 24 hours. mRNA expression of each organoid line was analyzed by qRT-PCR in duplicate. Each bar represents mean ± SEM.
Figure 4
Figure 4. Superposition of the three predicted MF binding modes.
Three different binding modes of MF to FXR, as determined by docking studies, are depicted. (A) Binding mode 1 suggests that the furoate group of MF is buried into the FXR binding site pointing towards helix 7 (yellow carbons); binding mode 2 is characterized by the furoate group of MF oriented toward helix 11 and helix 12 (blue carbons); binding mode 3 is head-to-tail flipped with respect to binding modes 1 and 2 with the furoate group of MF oriented toward the helix 5 and 6 (magenta carbons). Helix 12 is shown in red. (B) HEK293T cells transfected with NF-κB reporter, expression plasmids for wild type FXR (FXR-WT) or mutant FXR (FXR-W469A) and RXR, and pTK-renilla construct were treated with DMSO, 1 μM GW4064, 1 or 10 μM MF as indicated, or TNFα, for 24 hours. The NF-κB luciferase reporter assay was performed in quadruplicate. *p < 0.001 compared to FXR-W469A cells treated with GW4064 or MF plus TNFα.
Figure 5
Figure 5. Graphical representation of the MF-FXR interactions.
Graphical representation of the MF-FXR interactions and RMSD calculated during 100 ns of molecular dynamic simulations of the three binding modes suggested by docking studies. (A) binding mode 1 is stabilized mainly by hydrophobic interactions; (B) binding mode 2 is the most stabilized complex due to the high number of both hydrophobic and hydrophilic contacts conserved; (C) binding mode 3 is the less stabile binding mode despite conserved interactions are established during the simulation.
Figure 6
Figure 6. GW4064 and MF reduce p65 recruitment to pro-inflammatory gene promoters.
HepG2-GFP and HepG2-GFP-FXR cells were treated with DMSO, GW4064, MF, and TNFα, as indicated. Cross-linked chromatin from these cell lysates was precipitated using an anti-p65 antibody, and analyzed by qPCR with primers specific to the known p65 binding regions of CXCL2 and IL8.

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References

    1. Burris T. P. et al. Nuclear receptors and their selective pharmacologic modulators. Pharmacol Rev 65, 710–778 (2013). - PubMed
    1. Hollman D. A., Milona A., van Erpecum K. J. & van Mil S. W. Anti-inflammatory and metabolic actions of FXR: insights into molecular mechanisms. Biochim Biophys Acta 1821, 1443–1452 (2012). - PubMed
    1. Glass C. K. & Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nat Rev Immunol 10, 365–376 (2010). - PubMed
    1. Schacke H., Docke W. D. & Asadullah K. Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther 96, 23–43 (2002). - PubMed
    1. De Bosscher K., Haegeman G. & Elewaut D. Targeting inflammation using selective glucocorticoid receptor modulators. Curr Opin Pharmacol 10, 497–504 (2010). - PubMed

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