In this study we identified a set of genes that exhibited differential expression in the esophagus of treated, FP-responsive patients with EE compared with untreated subjects. Our analysis uncovered several genes previously shown to be glucocorticoid responsive, including those encoding FKBP51, MHC class II, and collagen molecules. Glucocorticoids upregulate FKBP51
, whereas they decrease expression of MHC class II and collagen genes in several cell types.16–19,21,22
The identification of such genes validates the analysis performed. Of the transcripts identified, all but 1 exhibited expression in nonresponders similar to that observed in untreated patients. This indicates that nonresponders might have a fundamental dysfunction in glucocorticoid signaling, at least in the esophagus. Tissue-specific glucocorticoid resistance has been reported in other inflammatory diseases, including rheumatoid arthritis, osteoarthritis, Crohn disease, ulcerative colitis, and asthma.23
Multiple mechanisms have been shown to account for glucocorticoid resistance, including aberrant interactions between transcription factors, coactivators, and corepressors; posttranslational modification of inflammatory mediators; and expression of glucocorticoid receptor isoforms.23
Interestingly, only 1 gene (F3
) showed differential expression in both FP responder and FP non-responders compared with untreated subjects. This implies that F3
gene expression could be influenced by a nongenomic mechanism of glucocorticoid signaling. This gene might be especially useful to monitor patient compliance because it is expressed in all patients who take the drug, regardless of responsiveness.
The observed change in patients’ biopsy specimen gene expression in addition to previous literature reporting a low systemic bioavailability of oral FP24
supports the interpretation that swallowed FP treatment exerts a topical effect in the esophagus. The oral bioavailability of FP has been reported to be less than 1% at a dose greater than 20-fold higher than the dose for patients with EE in this current study24
; this, coupled with the fact that FP undergoes extensive first-pass metabolism in the liver,25
makes it less likely that swallowed FP acts through a systemic route to alter gene expression in the esophagus. To our knowledge, this represents the first report suggesting direct evidence of a topical effect of glucocorticoids in the esophagus.
Here we report that FKBP51
was upregulated at the mRNA and protein levels in esophageal epithelial cells, including primary esophageal epithelial cells that were cultured from esophageal biopsy specimens. Based on the ability of RU486 to inhibit FKBP51 protein induction, the increased FKBP51 levels are likely glucocorticoid receptor dependent. We observed that increased transcript stability did not account for the upregulation of FKBP51
mRNA. Instead, increased FKBP51
mRNA levels are likely caused by increased transcription of the gene. Numerous putative glucocorticoid response elements (GREs) exist within the FKBP51
promoter and introns, and such sequences might mediate the increased transcription in cell types, including A549 cells.26
We observed that FKBP51
levels were increased at baseline on CHX treatment, suggesting that inhibition of protein synthesis up-regulates FKBP51
. This could occur if a repressor of FKBP51
transcription required ongoing protein synthesis; alternatively, FKBP51
levels might be upregulated as part of a stress response. Despite this increased baseline expression, FKBP51
transcription still increased on glucocorticoid treatment in the presence of CHX, suggesting that at least a portion of the increased FKBP51
transcripts do not require de novo
protein synthesis. These data collectively suggest that glucocorticoid signaling directly affects FKBP51
transcription in esophageal epithelial cells.
We observed that FP treatment inhibited IL-13–mediated eotaxin-3 promoter activity. This appears to be mediated through the 800 bp of promoter that contains 1 canonical GRE sequence. Previous reports have suggested that repression of IL-4–induced eotaxin-3 expression by FP occurs independently of this GRE sequence in lung epithelial cells.27
It remains to be tested whether this is the case in esophageal epithelial cells.
has been shown to act as a negative regulator of glucocorticoid signaling.28,29
In fact, baseline FKBP51
levels in airway epithelial cells negatively correlate with response to glucocorticoid treatment in asthmatic patients.30
Additionally, new world primates exhibit high levels of FKBP51
, and this correlates with general glucocorticoid resistance in these animals. 17,31,32
Herein, we show that increasing baseline FKBP51
levels in the esophageal cell line TE-7 correlate with a decreased ability of glucocorticoid to repress IL-13–mediated eotaxin-3 promoter activity ().
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) (more ...)
Biopsy specimens of patients who respond to swallowed FP treatment exhibit high levels of FKBP51
, a negative regulator of glucocorticoid signaling.28,29,31,32
However, we note that the observed high FKBP51
transcript levels occurred after treatment. In contrast, we showed that high baseline FKBP51
levels before treatment negatively affect glucocorticoid signaling in vitro
. Therefore we propose that although increased FKBP51
levels after FP treatment serve as a readout of functional glucocorticoid signaling, the actual biological function of FKBP51
in part involves negative regulation of glucocorticoid signaling to dampen the anti-inflammatory response so that homeostatic conditions can be restored after the glucocorticoid-mediated resolution of inflammation.
In summary, we have shown that (1) FP responders express a unique set of esophageal genes (including FKBP51), providing evidence that swallowed glucocorticoids mediate their role through a topical effect in the esophagus in vivo, and (2) FP nonresponders have a blunted expression of steroid-induced esophageal transcripts, suggesting impaired glucocorticoid signaling in these patients. Further proof for the ability of glucocorticoids to induce esophageal transcripts is derived by in vitro studies with esophageal epithelial cells, which have demonstrated that (1) glucocorticoids directly induce FKBP51 through a RU486-inhibited mechanism, indicating the direct involvement of the glucocorticoid receptor; (2) FKBP51 mRNA half-life is approximately 16 to 18 hours and importantly not regulated by glucocorticoids; and (3) FKPB51 overexpression inhibits glucocorticoid-mediated transcriptional repression of eotaxin-3. Taken together, our data provide molecular evidence that swallowed glucocorticoids mediate their action through a topical effect in the esophagus and that posttreatment FKBP51 esophageal levels serve as a measure of functional glucocorticoid signaling.
Clinical implications: Swallowed glucocorticoids induce disease remission by regulating local gene expression in the esophagus. Understanding the mechanism by which topical glucocorticoids induce remission from EE will provide insight into disease pathogenesis, steroid resistance, and additional molecular pathways to serve as targets for therapeutic intervention.