Glucocorticoid-regulated genes in eosinophilic esophagitis: a role for FKBP51

Abstract

Background: Eosinophilic esophagitis (EE) involves marked accumulation of eosinophils in the esophageal mucosa that responds to swallowed fluticasone propionate (FP) in a subset of patients.

Objectives: We aimed to uncover the mechanism of action of swallowed FP in patients with EE by providing evidence for a topical effect in the esophagus by identifying a molecular signature for FP exposure in vivo.

Methods: Global microarray expression profiles, immunofluorescence microscopy, and cell signaling in esophageal tissue and cell lines were analyzed.

Results: Thirty-two transcripts exhibited altered expression in patients who responded to swallowed FP treatment. Esophageal FK506-binding protein 5 (FKBP51) mRNA levels were increased (P < .05) in FP responders compared with those seen in control subjects and patients with untreated active EE. After FP treatment of esophageal epithelial cells, FKBP51 mRNA and protein levels were increased in a dose- and time-dependent manner by FP treatment in vitro. FP-induced FKBP51 was steroid receptor dependent because RU486 completely inhibited gene and protein induction. The half-life of FKBP51 mRNA was 16 to 18 hours independent of FP treatment. FKBP51 overexpression reduced FP action as assessed by FP inhibition of IL-13-induced eotaxin-3 promoter activity.

Conclusions: Our results suggest that swallowed glucocorticoid treatment directly affects esophageal gene expression in patients with EE. In particular, increased FKBP51 transcript levels identify glucocorticoid exposure in vivo and distinguish FP responders from untreated patients with active EE and patients without EE. In addition, FKBP51 reduces glucocorticoid-mediated inhibition of IL-13 signaling in epithelial cells in vitro, suggesting that FKBP51 might influence FP responsiveness. We propose that esophageal FKBP51 levels have diagnostic and prognostic significance in patients with EE.

FIG 1

Identification of glucocorticoid-regulated genes in patients with EE who respond to FP treatment.A, The average relative gene expression for each patient group.B,Average expression levels of transcripts identified in Fig 1, A. The scale is represented in Fig 1, A (right). C, Average expression of genes exhibiting increased transcript levels in FP responders. D, Average expression of genes showing decreased transcript levels in FP responders. EE, Untreated patients with EE; NL, control patients; NR, FP nonresponders; R, FP responders.

FIG 2

Verification of transcript levels of glucocorticoid-regulated genes by means of real-time PCR analysis. A, Average gene expression determined by means of microarray analysis is expressed as the fold change compared with that seen in control subjects. B, Transcript levels for the indicated gene were quantified by means of real-time PCR and normalized to GAPDH levels; samples used were collected from the same patients used for micro-array analysis. The graph displays the fold change compared with that seen in control subjects. EE, Untreated patients with EE; NL, control patients; NR, FP nonresponders; R, FP responders. *P < .05. **P < .01. ***P < .001.

FIG 3

Localization of FKBP51 expression in patients’ biopsy specimens. Esophageal biopsy sections were immunostained to visualize FKBP51 (red), and 4′-6-diamidino-2-phenylindole, dihydrochloride (DAPI) was used to visualize nuclei (blue). Magnification is ×200 for Fig 3, A and B, and ×800 for Fig 3, C. A, Esophageal biopsy specimen from an untreated patient with EE. B, Negative control (control antibody) for Fig 3, A. C, High-power magnification of biopsy specimen in Fig 3, A.

FIG 4

FKBP51 induction after glucocorticoid treatment of esophageal epithelial cells. For Fig 4, A, B, D, and E, protein extracts were subjected to SDS-PAGE and Western blot analysis for FKBP51 and actin; protein levels were quantified by means of densitometric analysis. The average fold change in the ratio of FKBP51 to actin for each sample compared with the untreated sample for 3 experiments is shown. For Fig 4, A through E, results are representative of 3 experiments. A, Primary esophageal epithelial cells were treated with 10⁻⁶ mol/L FP for 24 hours. B, TE-7 cells were treated with FP for 24 hours. C, TE-7 cells were treated with 10⁻⁷ mol/L FP. RNA was isolated, cDNA synthesis was performed, and real-time PCR analysis to detect FKBP51 and GAPDH transcripts was done. D, In a separate experiment TE-7 cells were treated for the indicated time with 10⁻⁷ mol/L FP. *P < .05. **P < .01. E, TE-7 cells were pretreated with 10⁻⁶ mol/L RU486 for 30 minutes. FP or dexamethasone (Dex) was then added for 24 hours. F, TE-7 cells were treated with 10⁻⁶ mol/L FP for 24 hours. Actinomycin D was then added (10 µg/mL). Cells were incubated for the indicated number of hours, at which time they were subjected to RNA isolation followed by cDNA synthesis. FKBP51 mRNA levels determined by means of real-time PCR were normalized to nanograms of reverse-transcribed RNA. The graph shows the ratio of FKBP51 per nanogram RNA as a percentage of the initial time point; each time point represents the mean ± SEM of 3 independent experiments. Half-life was calculated as the mean ± SEM of the half-life value for each of the 3 independent experiments. G, TE-7 cells were pretreated with 10 µg/mL CHX for 30 minutes. Subsequently, FP or dexamethasone was added for 24 hours. The graph shows FKBP51 mRNA expression normalized to GAPDH expression. The results are representative of 3 experiments. DMSO, Dimethyl sulfoxide.

FIG 5

FKBP51 transcript and protein levels are increased by dexamethasone (Dex) treatment of esophageal epithelial cells. A, RNA isolated from TE-7 cells treated with dexamethasone or FP for 24 hours was subjected to cDNA synthesis. FKBP51 transcript levels determined by means of real-time PCR analysis were normalized to GAPDH levels. B–D, Western blot analysis for FKBP51 and β-actin. Fig 5, B, TE-7 cells were treated with dexamethasone for 24 hours. Fig 5, C, TE-7 cells were treated with 10⁻⁶ mol/L dexamethasone. Fig 5, D, Primary esophageal epithelial cells were treated with 10^–6 mol/L dexamethasone for 24 hours. In Fig 5, A–D, data are representative of 3 experiments per figure.

FIG 6

IL-13–induced transcript and protein levels of eotaxin-3 are reversed by FP treatment of esophageal epithelial cells. A, RNA isolated from TE-7 cells treated with IL-13, FP, or both for 24 hours was subjected to cDNA synthesis. Transcript levels of eotaxin-3 were determined by means of real-time PCR analysis and normalized to GAPDH levels. B, TE-7 cells were treated with IL-13, FP, or both for 48 hours. ELISA was performed to detect eotaxin-3 in the supernatants. The dashed line indicates the detection limit of the assay. For Fig 6, A and B, data are representative of 3 experiments per figure. *P < .05. **P < .01.

FIG 7

Increased baseline FKBP51 levels affect glucocorticoid-mediated repression of IL-13–induced eotaxin-3 promoter activity. A, Constructs used in this study. CMV, Cytomegalovirus promoter. Cells were transfected with pHRL-TK and either pEotaxin-3 (B) or pEotaxin-3-3′UTR (C) or pHRL-TK, pEotaxin-3, and either pCDNA3.1 or pFKBP51 (D). Fig 7, B–D, After transfection, cells were treated with IL-13, FP, or both for 24 hours. Firefly and Renilla luciferase activities for each sample were then quantified. Eotaxin-3 promoter activity is expressed as the ratio of firefly to Renilla luciferase activity. For Fig 7, B–D, panels are representative of 3 experiments. ns, Nonsignificant. **P < .01. ***P < .001.

FIG 8

Model to describe the regulation and potential function of FKBP51 in patients with EE. FP represses IL-13–induced eotaxin-3 expression while inducing FKBP51 gene expression through a mechanism likely dependent on the glucocorticoid receptor (GR). FKBP51 additionally inhibits glucocorticoid receptor (GR)–mediated signaling and thus dampens glucocorticoid-mediated repression of IL-13–induced eotaxin-3 promoter activity.