Aberrant epithelial-mesenchymal Hedgehog signaling characterizes Barrett's metaplasia

Abstract

Background & aims: The molecular mechanism underlying epithelial metaplasia in Barrett's esophagus remains unknown. Recognizing that Hedgehog signaling is required for early esophageal development, we sought to determine if the Hedgehog pathway is reactivated in Barrett's esophagus, and if genes downstream of the pathway could promote columnar differentiation of esophageal epithelium.

Methods: Immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction were used to analyze clinical specimens, human esophageal cell lines, and mouse esophagi. Human esophageal squamous epithelial (HET-1A) and adenocarcinoma (OE33) cells were subjected to acid treatment and used in transfection experiments. Swiss Webster mice were used in a surgical model of bile reflux injury. An in vivo transplant culture system was created using esophageal epithelium from Sonic hedgehog transgenic mice.

Results: Marked up-regulation of Hedgehog ligand expression, which can be induced by acid or bile exposure, occurs frequently in Barrett's epithelium and is associated with stromal expression of the Hedgehog target genes PTCH1 and BMP4. BMP4 signaling induces expression of SOX9, an intestinal crypt transcription factor, which is highly expressed in Barrett's epithelium. We further show that expression of Deleted in Malignant Brain Tumors 1, the human homologue of the columnar cell factor Hensin, occurs in Barrett's epithelium and is induced by SOX9. Finally, transgenic expression of Sonic hedgehog in mouse esophageal epithelium induces expression of stromal Bmp4, epithelial Sox9, and columnar cytokeratins.

Conclusions: Epithelial Hedgehog ligand expression may contribute to the initiation of Barrett's esophagus through induction of stromal BMP4, which triggers reprogramming of esophageal epithelium in favor of a columnar phenotype.

Figure 1. Epithelial Hh activation in BE and esophageal adenocarcinoma

A-D) SHH immunohistochemistry. (40×) A) Esophageal squamous epithelium. B) BE. C) BE with high-grade dysplasia. D) Esophageal adenocarcinoma. E-F) SHH and IHH quantitative real-time PCR (qRT-PCR) data from 15 frozen esophagectomy cases. Note log scale of Y-axis. Absence of bar indicates undetectable expression levels.

Figure 2. Stromal Hh pathway targets are upregulated in BE

A) PTCH1 and B) BMP4 qRT-PCR data from 15 frozen esophagectomy cases. (C-F) Immunofluorescence performed on frozen esophagectomy cases. Nuclei stained with DAPI in blue. Anti-cytokeratin in green. C) BE and D) Achalasia stained with anti-PTCH1 in red. E) BE and F) Achalasia stained with anti-BMP4 in red. Insets are higher magnification of positive staining cells.

Figure 3. Physiologic causes of BE activate the Hh pathway

A) qRT-PCR data of HET-1A cells treated with non-acidified (pH 7.6) or acidified media for 48 hours. B) qRT-PCR data of mouse esophagi following esophagojejunostomy as compared to Swiss Webster whole esophagus. C) BE in Swiss Webster mouse 40 weeks after esophagojejunostomy. * BE between two regions of squamous epithelium. D) Hyperproliferative squamous epithelium in Swiss Webster mouse 40 weeks post-op. (20×) E-F) β-galactosidase activity in Ptch1-LacZ mouse 30 weeks post-op. (40×) E) Intestinal-like epithelium with positive blue staining in underlying stroma. F) Hyperproliferative squamous epithelium with absent staining.

Figure 4. SOX9 expression in normal human gastrointestinal tract, BE and esophageal adenocarcinoma

SOX9 immunohistochemistry. A) Esophagus. (40×) B) Stomach with staining in pit-gland transition zone. (20×) C) Duodenum with staining in intestinal crypts. (20×) D) BE. (40×) E) BE with high-grade dysplasia. (40×) F) Esophageal adenocarcinoma. (40×)

Figure 5. BMP4 signaling activates SOX9

A) SOX9 qRT-PCR data from 15 frozen esophagectomy cases. B) HET-1A cells treated with PBS (vehicle) or 100ng/ml BMP4 for three hours or transfected with pcDNA3.1 (empty vector) or BMPRIA* (constitutively active BMPRIA). qRT-PCR data for expression of SOX9 and the BMP target gene ID2. C) SOX9 immunocytochemistry in transfected HET-1A cells. (60×)

Figure 6. SOX9 activates DMBT1 expression

DMBT1 qRT-PCR data from A) 15 frozen esophagectomy cases and B) transfected HET-1A cells. C) SHH and SOX9 immunocytochemistry in OE33 cells. D) SOX9 Western blot with GAPDH loading control and E) DMBT1 qRT-PCR data of OE33 cells transfected with SOX9 siRNAs.

Figure 7. Shh signaling induces columnar differentiation in a 3-D tissue reconstitution model

Shh, Ck8/18, and Sox9 immunohistochemistry in 3-dimensional culture reconstituted from primary esophageal squamous epithelial cells isolated from conditional Shh-transgenic or wild-type littermate mice. Bars represent 20μm.

Figure 8. Proposed molecular model of metaplasia

1) Acid and bile injure esophageal squamous epithelium. 2) Injured epithelial cells secrete SHH and IHH which causes 3) mesenchymal secretion of BMP4. 4) BMP4 signals to epithelium activating SOX9. 5) SOX9 activates a columnar cell transcriptional program including DMBT1.