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. 2006 Mar 15;20(6):689-99.
doi: 10.1101/gad.1400506.

Novel arylpyrazole compounds selectively modulate glucocorticoid receptor regulatory activity

Jen-Chywan Wang  1 , Nilesh Shah, Carlos Pantoja, Sebastiaan H Meijsing, Joseph D Ho, Thomas S Scanlan, Keith R Yamamoto
Affiliations

"V体育安卓版" Novel arylpyrazole compounds selectively modulate glucocorticoid receptor regulatory activity

Jen-Chywan Wang et al. Genes Dev. .

Abstract

The activities of intracellular receptors are regulated by their cognate ligands VSports手机版. Here we show that a series of related arylpyrazole compounds, which specifically bind the glucocorticoid receptor (GR), selectively modulated GR-regulated biological functions in preadipocyte, pre-osteoblast, and lung epithelial cell lines. Indeed, when we monitored 17 endogenous GR target genes in one of these cell types, we found that distinct arylpyrazole compounds induced different expression patterns. We showed by chromatin immunoprecipitation that the arylpyrazole compounds regulated, in a gene-specific manner, either GR occupancy of the genomic glucocorticoid response element (GRE) or events after GR association, such as histone modification. Overall, our results establish that subtle differences in ligand chemistry can profoundly influence the transcriptional regulatory activity of GR, and that endogenous genes bearing natural GREs are especially sensitive detectors of these differences. .

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Figures (V体育官网入口)

Figure 1.
Figure 1.
Arylpyrazole compounds differentially affect cell growth and differentiation. (A) Effects of arylpyrazole compounds on A549 cell proliferation. A549 cells were treated with either DMSO, dexamethasone (0.1 μM), or arylpyrazole compounds (1 μM) for 5 d, and the relative ATP levels were measured. The data represent the mean (standard error of the mean, SEM) of the percent of ATP levels (dexamethasone or arylpyrazole compound-treated divided by the DMSO-treated) from four experiments. (B) Effects of arylpyrazole compounds on 3T3-L1 adipocyte differentiation. Confluent cultures of 3T3-L1 cells were placed in differentiation medium that contained IBMX, insulin with either DMSO, dexamethasone (1 μM), prednisolone (1 μM), or various arylpyrazole compounds (1 μM) as indicated. Oil Red staining was used to measure the extent of adipocyte differentiation. The data represent the mean (SEM) of Oil Red concentration from three experiments. (C) The effects of arylpyrazole compounds on MC3T3-E1 osteoblast differentiation. MC3T3-E1 cells were grown to confluence and then placed in differentiation medium that contained either DMSO, dexamethasone (1 μM), prednisolone (1 μM), or various arylpyrazole compounds (1 μM) as indicated. Alizarin Red staining was used to measure the extent of osteoblast differentiation. Data are averaged (with SEM) from four independent experiments.
Figure 2.
Figure 2.
Arylpyrazole compounds differentially affect the expression of GR target genes in A549 cells. (A) Effects of arylpyrazole compounds on dexamethasone-induced genes. A549 cells were treated with dexamethasone or various arylpyrazole compounds for 4–5 h. Total RNA was prepared and subjected to cDNA synthesis. The cDNA was then analyzed by qPCR to measure the relative mRNA levels of distinct target genes using gene-specific primers. Red bars represent induction greater than threefold. Orange represents induction between two- and threefold. Yellow represents induction between 1.5-fold and twofold. (B) Effects of arylpyrazole compounds on TNFα-induced dexamethasone-suppressed genes. A549 cells were treated with TNFα and either dexamethasone or arylpyrazole compounds for 4 h. Total RNA was prepared and subjected to cDNA synthesis. The cDNA was then analyzed by qPCR to measure the relative mRNA levels of distinct target genes using gene-specific primers. Green represents percent inhibition >50%. Medium green represents percent inhibition between 25% and 50%. Light green represents percent inhibition between 0% and 25%. (C) Effects of arylpyrazole compounds on dexamethasone-suppressed genes. A549 cells were treated with TNFα and dexamethasone or various arylpyrazole compounds for 4 h. Total RNA was isolated and subjected to cDNA synthesis. The cDNA was then analyzed by qPCR to measure the mRNA levels of distinct target genes. The representative colors are as described above. The data in A–C represent the mean of the fold induction (DEX-treated responses divided by the DMSO-treated responses) from at least three experiments. The SEM is shown in the Supplemental Material. (D) The effects of arylpyrazole compounds on reporter genes containing simple GRE. Seventy-five nanograms of TAT3 reporter plasmids were transfected with 100 ng of RSV-βGal into A549 cells in a 24-well plate. After 24 h, cells were washed with PBS and treated with 0.1 μM DEX for an additional 16–20 h. Cells were then lysed and subjected to assays for luciferase and β-Gal activities. One representative data set from three independent transfection experiments is shown.
Figure 3.
Figure 3.
Ligands 13 and 15 induce GR nuclear localization. Subconfluent eGFP–rGR cells were treated with DMSO (vehicle control) (A), 100 nM dexamethasone (B), 1 μM ligand 13 (C), and 1 μM ligand 15 (D) for 4 h. Localization of eGFP–rGR protein was then monitored. Representative data from two experiments are shown.
Figure 4.
Figure 4.
Ligand 15 inhibits prednisolone-regulated cell proliferation and gene expression in A549 cells. (A) A549 cells were treated as indicated for 4–5 h. Total RNA was isolated and subjected to cDNA synthesis. The cDNA was then analyzed by qPCR to measure mRNA levels of distinct GR target genes: GILZ (solid bar), Kip2 (open bar), and MCJ (hatched bar). The data represent the mean (SEM) of the fold induction (relative to DMSO-treated responses) from four experiments. (B) A549 cells were treated with distinct ligands as indicated for 5 d. The relative ATP levels of cells were then measured. The data represent the mean (SEM) of the fold induction (relative to DMSO-treated responses) from three experiments.
Figure 5.
Figure 5.
Arylpyrazole compounds differentially affect GR occupancy at distinct GREs. A549 cells were treated with prednisolone or various arylpyrazole compounds for 1 h, and ChIP experiments were performed to assess occupancy by GR. The results for the following genes are shown: ENaCα (A), GILZ (B), and SLC19A2 (C). The fold enrichment values for the experimental regions are determined by normalizing to the control hsp70 value. The data represent the mean (SEM) of the fold enrichment (relative to DMSO-treated responses) from at least three experiments.
Figure 6.
Figure 6.
Prednisolone but not ligand 15 increases histone acetylation at the ENaCα gene. (A) Schematic diagram of the ENaCα gene; closed boxes indicate exons, and the transcription start site is indicated as +1. Amplified genomic regions are underlined and numbered. (B) A549 cells were treated with prednisolone (1 μM) or ligand 15 (1 μM) for 30 min, and actylated histone H3 was measured in each numbered region by ChIP. (C) A549 cells were treated with prednisolone (1 μM) or ligand 15 (1 μM) for 30 min, and actylated histone H4 was measured in each numbered region by ChIP. Fold enrichment values in B and C were determined by normalizing to a control gene, hsp70. The data represent the mean (SEM) of the fold enrichment (relative to DMSO-treated responses) from at least three experiments.
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