Omeprazole blocks STAT6 binding to the eotaxin-3 promoter in eosinophilic esophagitis cells

Abstract

Background: Patients who have esophageal eosinophilia without gastroesophageal reflux disease (GERD) nevertheless can respond to proton pump inhibitors (PPIs), which can have anti-inflammatory actions independent of effects on gastric acid secretion. In esophageal cell cultures, omeprazole has been reported to inhibit Th2 cytokine-stimulated expression of eotaxin-3, an eosinophil chemoattractant contributing to esophageal eosinophilia in eosinophilic esophagitis (EoE). The objective of this study was to elucidate molecular mechanisms underlying PPI inhibition of IL-4-stimulated eotaxin-3 production by esophageal cells.

Methods/findings: Telomerase-immortalized and primary cultures of esophageal squamous cells from EoE patients were treated with IL-4 in the presence or absence of acid-activated omeprazole or lansoprazole. We measured eotaxin-3 protein secretion by ELISA, mRNA expression by PCR, STAT6 phosphorylation and nuclear translocation by Western blotting, eotaxin-3 promoter activation by an exogenous reporter construct, and STAT6, RNA polymerase II, and trimethylated H3K4 binding to the endogenous eotaxin-3 promoter by ChIP assay. Omeprazole in concentrations ≥5 µM significantly decreased IL-4-stimulated eotaxin-3 protein secretion and mRNA expression. Lansoprazole also blocked eotaxin-3 protein secretion. Omeprazole had no effect on eotaxin-3 mRNA stability or on STAT6 phosphorylation and STAT6 nuclear translocation. Rather, omeprazole blocked binding of IL-4-stimulated STAT6, RNA polymerase II, and trimethylated H3K4 to the eotaxin-3 promoter.

Conclusions/significance: PPIs, in concentrations achieved in blood with conventional dosing, significantly inhibit IL-4-stimulated eotaxin-3 expression in EoE esophageal cells and block STAT6 binding to the promoter. These findings elucidate molecular mechanisms whereby patients with Th2 cytokine-driven esophageal eosinophilia can respond to PPIs, independent of effects on gastric acid secretion.

Figure 1. Omeprazole decreases IL-4-stimulated eotaxin-3 protein secretion in primary esophageal squamous cells from two patients with EoE, (A) EoE1 and (B) EoE2.

Data are the mean ± SEM of 2 separate experiments. ***, p≤0.001 compared to unstimulated (baseline) control, ⁺⁺, p≤0.01 compared to IL-4 stimulation alone; ⁺⁺⁺, p≤0.001 compared to IL-4 stimulation alone. Unstim.; unstimulated cells.

Figure 2. Omeprazole decreases IL-4-stimulated eotaxin-3 protein secretion intelomerase-immortalized esophageal squamous cell lines from two patients with EoE, (A) EoE1-T and (2) EoE2-T.

Data are the mean ± SEM of 2 separate experiments. ***, p≤0.001 compared to unstimulated (baseline) control, ⁺⁺⁺, p≤0.001 compared to IL-4 stimulation alone. Unstim.; unstimulated cells.

Figure 3. Lansoprazole decreases IL-4-stimulated eotaxin-3 protein secretion in (A) EoE1-T and (2) EoE2-T cells.

Data are the mean ± SEM of at least 2 separate experiments. ***, p≤0.001 compared to unstimulated (baseline) control, ⁺⁺⁺, p≤0.001 compared to IL-4 stimulation alone. Unstim., unstimulated cells.

Figure 4. Omeprazole (Ome) decreases IL-4-stimulated eotaxin-3 mRNA expression levels in (A) EoE1-T and (B) EoE2-T cell lines as determined by conventional PCR and quantitative real-time PCR.

Depicted is of one of at least 2 separate experiments. **, p≤0.01 compared to IL-4 stimulation alone; ***, p≤0.001 compared to IL-4 stimulation alone. C, unstimulated control cells.

Figure 5. Omeprazole (Ome) does not increase IL-4-stimulated eotaxin-3 mRNA degradation, and does not decrease STAT6 phosphorylation or nulcear translocation in EoE1-T and EoE2-T cell lines.

mRNA expression levels were determined by quantitative real-time PCR in (A) EoE1-T and (B) EoE2-T cells. Data are the means ± SEM of 3 separate experiments assayed in triplicate. Representative experiments of Western blotting for (C) phospho- and total STAT6 in whole cell lysates and (D) phospho-STAT6 in nuclear lysates in EoE1-T and EoE2-T cells. Tubulin and lamin A/C served as controls for the whole cell and nuclear lysates, respectively. Unstim., unstimulated cells.

Figure 6. Omeprazole (Ome) decreases IL-4-stimulated STAT6 binding to the endogenous eotaxin-3 promoter in (A) EoE1-T and (B) EoE2-T cells.

Data are the means ± SEM of 3 separate experiments. ***, p≤0.001 compared to IL-4 stimulation. Isotype matched IgG served as a control. Omeprazole does not inhibit activation of the transiently transfected, exogenous, eotaxin-3 EO1 promoter (−800 bp) construct in (C) EoE1-T and (D) EoE2-T cells. Data are the mean ± SEM of 2 separate experiments. ***, p<0.001 compared to unstimulated control cells.

Figure 7. Omeprazole (Ome) decreases IL-4-stimulated RNA Pol II binding to the endogenous eotaxin-3 promoter in (A) EoE1-T and (B) EoE2-T cells.

Data are the means ± SEM of 3 separate experiments. **, p≤0.01 compared to IL-4 stimulation; ***, p≤0.001 compared to IL-4 stimulation Isotype matched IgG served as a control. Representative experiment demonstrating that omeprazole reduces the levels of IL-4 stimulated H3K4me3 bound to the endogenous eotaxin-3 promoter in (C) EoE1-T and (D) EoE2-T cells. Isotype matched IgG served as a control. Depicted is one of 3 separate experiments. M, marker.