Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa

Abstract

Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of Barrett's metaplasia (BM), with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-κB, driving the aberrant expression of intestine-specific caudal-related homeobox (CDX) genes. However, early events in the pathogenesis of BM from normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-κB activation without causing cell death. Informed by this, the 3D oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and Metacore™; GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-κB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4α were highlighted. Our data suggest that HNF4α isoform switching may be an early event in Barrett's pathogenesis. CDX1/2 targets were, however, not enriched, suggesting that although CDX1/2 activation reportedly plays a role in BM development, it may not be an initial event. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets.

Keywords: Barrett's metaplasia; HNF4alpha; acid; oesophageal adenocarcinoma; reflux; taurochenodeoxycholate; tissue engineering.

Figure 1

Effect of bile salts on metabolic activity of HET-1A cells. Cells were subjected to pulsatile exposure to individual bile salts at pH 7.4 and pH 4. Metabolic activity was measured as a percentage of control cells treated with pH 7.4 media. Differences were calculated between treatments and control by one-way anova and Bonferroni post-test. Significant decreases in metabolic activity are indicated by **(P < 0.01) and ****(P < 0.0001).

Figure 2

The effect upon (a) total levels of NF-κB and (b) NF-κB localization from pulsatile exposure of HET-1A cells to bile salts in the absence and presence of acid. Results from all treatments were compared to the pH 7.4 control using anova with Bonferroni post-test. Statistical significance is indicated by **(P < 0.01), ***(P < 0.001) and ****(P < 0.0001). Results from bile treatment at pH 4 were also compared to the pH 4 only control using anova with Bonferroni post-test (shown in grey *P < 0.05).

Figure 3

Venn diagram with values indicating the number and extent of overlap for the differentially expressed genes in the epithelium of the oesophageal model following pulsatile exposure to acid and/or taurochenodeoxycholate compared to the control. Results are given for posterior probability of positive log ratio <0.3 or >0.7.

Figure 4

The broad Gene Ontology categories significantly represented by the dysregulated gene lists following pulsatile exposure of the model oesophagus to acid and/or taurochenodeoxycholate.

Figure 5

The concentrations of the alternative part of the 5′UTR of HNF4α, exon 1, which is driven by P1 promoter relative to the control, determined by qRT-PCR. Results are normalized against the pH 7.4 treated control.

Figure 6

H + E staining of organotypic models following 2 weeks pulsatile (2 × 10 min per day) exposure to taurochenodeoxycholate (TCDC) +/− acid. (a) was exposed to pH 7.4, (b) was treated with TCDC, (c) received pH 4 treatment and (d) pH 4 and TCDC.

Figure 7

HNF4 immunohistochemical staining of clinically defined Barrett's metaplasia (BM) and adjacent normal oesophageal squamous epithelia (left) and composites (right). The antibodies used were anti-HNF4α/γ (a, b), anti-HNF4γ (c, d), anti-P1-HNF4α, specific for isoforms 1–4 (e, f) and anti- P2-HNF4α, specific for isoforms 7–9 (g, h and i). Regions of metaplasia (BM) and normal squamous epithelium (N) within the tissue are labelled. Where both BM and normal tissue are present in the same image, the BM boundary is indicated with a grey dotted line.