Digestive and Liver Disease
Volume 43, Issue 1 , Pages 54-59, January 2011

Expression of Sonic hedgehog (SHH) and CDX2 in the columnar epithelium of the lower oesophagus

  • Yoshiyuki Yamanaka

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • Corresponding Author InformationCorresponding author at: 577 Matsushima, Kurashiki City, Okayama Prefecture 701-0192, Japan. Tel.: +81 86 462 1111; fax: +81 86 464 1195.
    • ,
  • Akiko Shiotani

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Yoshinori Fujimura

      Affiliations

    • Department of Internal Medicine, Center for Gastroenterology and Endoscopy, Kawasaki Hospital, Kawasaki Medical School, Okayama, Japan
    • ,
  • Manabu Ishii

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Minoru Fujita

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Hiroshi Matsumoto

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Ken-ichi Tarumi

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Tomoari Kamada

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan
    • ,
  • Jiro Hata

      Affiliations

    • Department of Clinical Pathology and Laboratory Medicine, Kawasaki Medical School, Japan
    • ,
  • Ken Haruma

      Affiliations

    • Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, Japan

Received 16 November 2009; accepted 24 April 2010. published online 09 July 2010.

Article Outline

Abstract 

Background

Decreases in Sonic hedgehog (SHH) and CDX2 expression are associated with atrophy and intestinal metaplasia in the gastric mucosa. The pathogenesis of development of Barrett's oesophagus is still unclear.

Objective

To examine the gene expression of CDX2 and SHH and their signalling pathways in the columnar epithelium and the association with endoscopic appearance, gastric pH or bile acids.

Subjects/Methods

Sixty-three patients with metaplastic columnar epithelium of the lower oesophagus were studied. Whole biopsy specimens and microdissected tissues were examined for messenger RNA.

Results

BMP4 expression was significantly higher in patients with tubular mucosal patterns of columnar epithelium visualised by Narrow Band Imaging with magnification. The expression of SHH was significantly lower and that of CDX2 was higher in the goblet columnar epithelium than in non-goblet columnar epithelium. CDX2 expression was significantly higher in the patients with hypoacidity than in the others. BMP4 and PTCH1 expression was significantly higher in the group with higher concentrations of deoxycholic acid than in the group with lower concentrations.

Conclusions

SHH might be the initial factor inducing columnar metaplasia, and subsequent or simultaneous BMP4 stimuli might induce the CDX2 expression that causes goblet-cell metaplasia.

Keywords: BMP4, CDX2, Columnar epithelium, SHH

 

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1. Introduction 

The majority of oesophageal adenocarcinomas evolve through a multistep process starting with conversion of squamous epithelium to mucinous columnar epithelium and progressing through goblet-cell metaplasia followed by the development of dysplasia and finally carcinoma [1], [2], [3]. This is true of intestinal metaplasia, which is recognised histologically by the presence of goblet cells, and is thought to be a precancerous lesion along the multistep path to gastric carcinoma [4], [5]. Barrett's oesophagus is generally defined by the presence of metaplastic columnar epithelium, which consists of a variety of cell types, including goblet and non-goblet mucinous columnar cells [6]. The traditional definition of Barrett's oesophagus included the presence of columnar epithelium of 3cm or greater in the distal oesophagus [7]. Previous studies evaluating the association of oesophageal adenocarcinoma with Barrett's oesophagus have only included patients with traditional or long-segment Barrett's oesophagus. However, recent studies have suggested that dysplasia and adenocarcinoma can also be associated with short-segment Barrett's oesophagus (SSBE), i.e., less than 3cm of columnar epithelium [8], [9]. A recent review provides evidence to suggest that a diagnosis of Barrett's oesophagus should not require demonstration of goblet cells in mucosal biopsies [10]. Emerging evidence suggests that non-goblet columnar epithelium shows characteristics of intestinal differentiation and demonstrates molecular abnormalities that indicate a risk of neoplastic change [11]. Although the molecular pathogenesis of Barrett's oesophagus is poorly understood, recent reports have indicated that dedifferentiation of squamous epithelium is one of the mechanisms of conversion to columnar epithelium [12].

The CDX proteins are intestine-specific transcription factors encoded by the CDX1 and CDX2 genes, which are the mammalian homologues of the Drosophilia homeobox gene, caudal [13], [14]. Aberrant expression of CDX2 in the upper gastrointestinal (GI) tract is thought to be a key event in the pathogenesis of Barrett's mucosa in the oesophagus as well as in intestinal metaplasia in the stomach [15], [16]. On the other hand, Sonic hedgehog (SHH) is a peptide morphogen which was initially identified during genetic screening of the fruit fly Drosophila [17], and is abundantly expressed in the normal gastric fundus. Ectopic SHH expression has been observed in fundic gland metaplasia [18], [19] including Barrett's oesophagus, whereas no SHH protein was observed in the normal oesophagus. SHH null mice exhibit GI malformations with replacement of the normal gastric epithelium with intestinal metaplastic cells [20], [21]. SHH binds to its receptor ‘Patched’ (PTC-PTCH1, PTCH2), which reduces the inhibitory effect of PTCH on another transmembrane protein, ‘smoothened’ (SMO). Binding results in de-repression of SMO, which activates a cascade that leads to the translocation of the active form of the transcription factor glioma-associated oncogene homologue (GLI) to the nucleus [22], [23], [24]. Hh signalling activation leads to cell proliferation through cell cycle regulation. Nuclear GLI activates the expression of a variety of target genes including BMP4 [18], [25], [26].

However, there have been few studies investigating SHH expression and its downstream signal in metaplastic columnar epithelium.

In the present study, we examined the gene expression of CDX2 and SHH and their signalling pathways in the non-goblet and goblet columnar epithelium of the lower oesophagus and the association of expression of these genes with endoscopic appearance or clinical characteristics. We also attempted to investigate the association of their expression with gastric pH and bile acids.

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2. Subjects and methods 

The subjects of this study were outpatients with metaplastic columnar epithelium of the lower oesophagus (segment length >1cm) detected by previous endoscopy who were recommended prior to upper GI endoscopy following reflux oesophagitis, screening of ulcers or cancer regardless of GI symptoms. The study was performed at Kawasaki Medical School Hospital in Japan and was approved by the Kawasaki Medical School Ethical Committee. Informed consent was obtained from each patient, and the patients were enrolled in the study between January 2007 and December 2008.

All subjects underwent upper GI endoscopy. Exclusion criteria were the taking of non-steroidal anti-inflammatory drugs (NSAIDs) and/or a history of gastrectomy. Patients were also excluded if they had gastric cancer or other malignant lesions, haemorrhagic diseases, insulin-dependent diabetes mellitus, cirrhosis or renal failure. Demographic data collected at study entry included age, sex, smoking habits, alcohol consumption and drug treatments including the use of anti-secretory drugs. Drinking and smoking were defined as regular intake when consumption was more than 35g of ethanol or five cigarettes per day, respectively.

2.1. Endoscopic examination 

All endoscopies were performed with an ETMI system using zoom endoscopy (GIF-Q240Z or GIF-H260Z, Olympus Inc., Tokyo, Japan). The light source (XCLV-260HP) contains two rotating red–green–blue RGB filters; one conventional for high-resolution white-light endoscopy (WLE) and an additional one for Narrow Band Imaging (NBI) in which the band-pass ranges are narrowed to wavelengths of 530–550nm (green) and 390–445nm (blue). Endoscopies were performed by experienced endoscopists using WLE and NBI with magnification (NBI-Z) after patients had fasted for 12h, and endoscopic photographs were obtained for subsequent review by two well-trained endoscopists. Magnified observation of the lower oesophagus using NBI-Z to determine the pit patterns of columnar epithelium which were classified into three patterns; small round, long oval and tubular (Fig. 1) [27]. Two specimens, one to be used as a whole sample and the other for laser-captured microdissection, were taken from 1cm proximal side from the oesophagogastric junction (EGJ) using endoscopic forceps (FB231K (A) Olympus). Squamous epithelium was taken from 1cm proximal side from the squamocolumnar junction (SCJ). The biopsy samples were immediately frozen using liquid nitrogen and stored at −80°C until use.

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  • Fig. 1. 

    Endoscopic images of lower oesophagus with metaplastic columnar epithelium by high-resolution white-light endoscopy (WLE) and three pit patterns by narrow band imaging with magnification (small round, long oval, and tubular).

2.2. Laser-captured microdissection 

Frozen samples were embedded in TissueTek OCT medium (VWR Scientific, Torance, CA). Cryostat sections (8μm) were laser-microdissected with a PixCell II laser-microdisecction system (Arcturus Engineering, Mountain View, CA).

2.3. RNA extraction and quantitative polymerase chain reaction 

Total RNA was extracted from whole biopsy samples using the RNeasy Mini kit (Qiagen, Hilden, Germany) and from microdissected tissue using a Pico Pure RNA Isolation kit (Arcturus Bioscience). Then cDNA synthesis was performed using the SuperScriptIII First Strand Synthesis System (Invitrogen, Carlsbad, CA, US). Quantitative reverse transcription (RT)-PCR analysis of SHH, PTCH1, GLI1, BMP4, CDX2, MUC5AC, MUC6, MUC2 and β-actin mRNA expression was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) employing the TaqMan gene expression assay according to the manufacturer's instructions (Applied Biosystem). The RT-PCR was performed with 20ng RNA for both target genes and the endogenous control using the TaqMan one-step RT-PCR Master Mix Reagent kit (P/N 4309169). Each amplification reaction was performed in triplicate, and the average of the threshold cycles was used. The target amount was obtained by normalisation to an endogenous reference (β-actin) and relative to a calibrator.

2.4. Immunohistochemistry 

Immunohistochemical staining for SHH and CDX2 was performed with the Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA, USA) using previous methods [28], [29]. Two adjacent 4μm thick sections were cut onto polylysine-coated glass slides. The sections were incubated with goat polyclonal SHH antibody (1:100, Santa Cruz, CA, USA) or mouse monoclonal CDX2 antibody (1:500, BioGenex, San Ramon, CA, USA) overnight at 4°C.

2.5. H. pylori diagnosis 

H. pylori status was determined by the presence of serum H. pylori IgG antibodies with an enzyme-linked immunosorbent assay (ELISA) kit using the E plate test (Eiken Kagaku, Inc., Tokyo, Japan).

2.6. Assay for bile acid in the gastric juice 

More than 1mL of gastric juice was endoscopically collected from the stomach, and samples were immediately stored at −20°C until use. Total bile acids of the gastric juice were measured by an enzyme method and the fraction of bile acid was investigated by HPLC method.

2.7. Statistical analyses 

Values are expressed as the mean±SD or the median with a 25–75% range, whichever was appropriate depending on whether the data were normally distributed. Statistical analyses for significant differences of parameters were performed using the non-parametric Mann–Whitney U test between the two groups. A two-sided p value of less than 0.05 was considered statistically significant. All statistical computations were performed using SPSS (SPSS Inc., Chicago, IL, USA).

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3. Result 

The study group consisted of 63 patients (44 men, 19 women, average age 64 years) with an endoscopic diagnosis of metaplastic columnar epithelium of the lower oesophagus. The segment length of the columnar epithelium in the all patients was less than 3cm. Demographic and clinical characteristics of the study group are shown in Table 1.

Table 1. Clinical characteristics of the patients.
Age, mean (SD)63.9 (14.3)
Gender, men (%)44 (69.8)
Drinking (%)25 (39.7)
Smoking (%)14 (22.2)
Antacid (%)a18 (28.5)
Eradication of H. pylori (%)11 (17.4)

aWe had no detailed information about antacid for one patient. Thirteen patients were taking proton pump inhibitor and four patients were taking H2 receptor antagonist.

3.1. Whole biopsy samples 

In the classification of columnar epithelium using magnified NBI observation, the frequencies of small round, long oval and tubular patterns were 11%, 49% and 40%, respectively. BMP4 expression was significantly higher (1.01×10−3 vs. 0.44×10−3, p=0.03) in the patients with the tubular mucosal pattern of columnar epithelium visualised by NBI-Z than in those with the long oval pattern combined with or without the small round pattern.

SHH, MUC5AC and MUC6 were expressed in the columnar epithelium in all subjects, and CDX2 and MUC2 in the columnar epithelium were detected in 77% and 72% of the subjects, respectively. However, there was no expression of SHH and CDX2 in the squamous epithelium of the oesophagus. SHH expression levels correlated with MUC5AC (r=0.72, p<0.001), while CDX2 levels correlated with MUC2 (r=0.84, p<0.001).

Gastric juice was collected from 54 of 63 subjects, and the median pH was 6.0. CDX2 expression was significantly higher (3.05×10−3 vs. 0.38×10−3, p=0.009) in patients with hypoacidity (gastric juice pH 6 or more) than in the other patients (Fig. 2). However, expression of other genes, including SHH did not differ between the two groups.

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  • Fig. 2. 

    Comparisons of CDX2 expression in the metaplastic columnar epithelium between the patients with hypoacidity (gastric juice pH 6 and >6) and the other (pH <6). Horizontal bar=median; box=25th–75th interquartile range; vertical lines=range of values. p values were calculated using the non-parametric Mann–Whitney U test. Data are expressed relative to the control gene β-actin.

MUC6 expression was significantly lower (160×10−3 vs. 591×10−3, p=0.01) in the group with higher concentrations of total bile acids (10 and >10μmol/L) in gastric juice than in the group with a lower concentration. Expression of any other gene did not differ significantly between the two groups. In contrast, the expression of BMP4 and PTCH1 was significantly higher (20.9×10−3 vs. 0.41×10−3, p=0.02 and 133×10−3 vs. 9.5×10−3, p=0.03) in the group with higher concentrations of deoxycholic acid (0.1 and >0.1μmol/l) than in the other group (Fig. 3).

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  • Fig. 3. 

    Comparisons of BMP4 and PTCH 1 expression between the patients with higher concentrations of deoxycholic acid (>0.1μmol/L) in gastric juice and the other patients (<0.1μmol/L). Horizontal bar=median; box=25th–75th interquartile range; vertical lines=range of values. p values were calculated using the non-parametric Mann–Whitney U test. Data are expressed relative to the control gene β-actin.

There was no significant association between the expression of each gene in the columnar epithelium and clinical characteristics including H. pylori infection.

3.2. Microdissected goblet and non-goblet columnar epithelium 

The levels of SHH (4.5×10−3 vs. 83×10−3, p=0.003), PTCH1 (5.2×10−3 vs. 54×10−3, p=0.002), and MUC5AC (1274×10−3 vs. 7178×10−3, p=0.03) were significantly lower in the goblet columnar epithelium than in the non-goblet columnar epithelium. CDX2 expression (11.7×10−3 vs. 0.5×10−3, p=0.08) tended to be higher and that of MUC2 (349 vs. 0, p=0.03) and was significantly higher in the goblet columnar epithelium than in the non-goblet columnar epithelium (Table 2).

Table 2. Comparisons of gene expression in non-goblet columnar epithelium and that in goblet columnar epithelium.
Non-goblet columnar epithelium median (25–75%)×10−3Goblet columnar epithelium median (25–75%)×10−3p
CDX20.47 (0–3.44)11.7 (1.89–19.6)0.08
SHH82.8 (29.8–158)4.45 (0.94–17.5)0.003
PTCH154.2 (21.2–108)5.24 (0.31–10.4)0.002
BMP41.45 (0–4.39)1.35 (0.14–38.0)0.63
MUC20 (0–8.7)349 (81.6–585)0.03
MUC5AC7178 (4593–17011)1274 (116–6024)0.03

Values are expressed relative to the control gene β-actin as the median with a 25–75% range. p values were calculated using the non-parametric Mann–Whitney U test.

3.3. Immunohistochemical staining 

SHH-positive staining was observed in the non-goblet glands but not in the goblet glands. CDX2 was expressed in the goblet columnar epithelium (Fig. 4).

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  • Fig. 4. 

    Sonic hedgehog (SHH) and CDX2 immunohistochemical staining in the metaplastic columnar epithelium (original magnification ×40). SHH-positive staining was observed in the non-goblet glands but not in the goblet columnar epithelium (a). CDX2 was expressed in the goblet columnar epithelium (b).

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4. Discussion 

In the present study, SHH and PTCH1 were expressed in almost all columnar epithelium and GLI was expressed as frequently as the CDX2 gene. By contrast, those genes were not detected in the squamous epithelium. The expression of SHH and gastric mucin was significantly lower and that of intestinal mucin MUC2 and CDX2 was higher in the columnar mucosa with goblet cells than in that without goblet cells shown by the results of immunostaining. These results confirm previously reported results suggesting that aberrant CDX2 and SHH are highly expressed in Barrett's epithelium and may play a crucial role in the development of Barrett's epithelium [30]. The expression patterns of these genes in the columnar mucosa with goblet cells were similar to those in gastric glands with intestinal metaplasia [28], [29].

BMP4 was expressed in the most of the columnar epithelium of our subjects. BMP4 is a protein belonging to the transforming growth factor (TGF)-β family, which is involved in controlling cellular differentiation, migration, and proliferation [12], [31]. SHH and BMP4 are presumably highly expressed in the mesenchyme of the oesophagus during early embryogenesis, but it seems that their action is closely balanced through simultaneous expression of the inhibitory noggin.

In a previous serial analysis of gene expression (SAGE) analysis, BMP4 was found to be abundantly and uniquely expressed in metaplastic columnar epithelium but not in normal squamous or cardia epithelium as in the present results [32], [33]. In another remarkable recent finding, a comparison of the gene expression profiles of squamous cells, BMP4-treated squamous cells and Barrett's epithelium cells showed a significant shift in the profile of the BMP4-treated squamous cells towards that of the cultured Barrett's epithelium cells [34].

In vitro studies previously demonstrated that bile acids and gastric acid exposure activated the CDX promoter in certain oesophageal cell lines and acid stimulation induced transcription of SHH in a gastric cancer cell line [35], [36]. However, in our present in vivo study analysing gastric aspirates, CDX2 gene expression was significantly higher in the patients with hypoacidity (gastric juice pH >6), while no indifferences were observed in other genes between the patients with hypoacidity and the others. We could not confirm associations between the acid stimulation and genes expression in the metaplastic columnar epithelium. This may have been because we investigated the gastric aspirates instead of performing 24h oesophageal pH monitoring and included the patients taking antacids and H. pylori-positive or eradicated patients. A strong direct correlation has been reported between the concentration of bile acids, and particularly unconjugated bile acids, in patients with GERD with a degree of oesophageal mucosal damage. This highlights the importance of refluxate components other than acid in oesophageal cancer progression [37], [38]. Bile acid in combination with a low pH is known to induce oxidative stress and oxidative DNA damage. However, a recent paper described the protective effects of glycoursodeoxycholic acid in Barrett's oesophagus cells [39].

Interestingly, the expression of BMP4 and PTCH1 was significantly higher in patients with higher concentrations of deoxycholic acid (>0.1μmol/l) than in other patients, although MUC6 gene expression was significantly lower in those with higher concentrations of total bile acids in gastric juice. Deoxycholic acid is a toxic unconjugated bile acid formed by the action of bacteria on primary bile acid. Although it is unclear which factors induce BMP4 expression in Barrett's epithelium, it is possible that both SHH and BMP4 play essential roles in the process of cellular development and differentiation [12].

A number of endoscopic methods including chromoendoscopy [40], [41], [42], [43] have been tried for the optical detection of intestinal metaplasia in the oesophagus and stomach [43]. Endo et al. classified the fine mucosal patterns (pit patterns) of Barrett's mucosa into five categories (small round, straight, long oval, tubular, and villous) by magnifying observation. The tubular and villous pit patterns were characteristics of the presence of goblet cells possessing an intestinal mucin phenotype. The long oval pit pattern had an intermediate phenotype between gastric and intestinal, whereas the other patterns possessed no goblet cells [27]. However, in the present study, using magnified NBI observation, which has been proven to improve the detection of pre-malignant and early neoplastic lesions, there were no significant differences in the gene expression except for that of BMP4. BMP4 expression was significantly higher in the patients with the tubular mucosal pattern visualised by NBI-Z than that in the other patterns. The tubular mucosal pattern might be characteristic of progressive Barrett's oesophagus and might be a risk marker for adenocarcinoma. The fact that no differences in other genes mRNA expression at the mucosa exhibiting the tubular mucosal pattern and the other patterns were observed might have been due to the small number of subjects and/or to sampling errors because the segments taken were too small.

In summary, the expression of SHH and gastric mucin was significantly lower and that of intestinal mucin and CDX2 was higher in the columnar mucosa with goblet cells than in that without goblet cells. These patterns were similar to those in gastric glands with intestinal metaplasia in patients with a high risk for gastric cancer. We found a significant association between BMP4 expression with a mucosal appearance by magnified NBI observation and a high concentration of deoxycholic acid in gastric juice. SHH might be the initial factor inducing columnar metaplasia, and subsequent or simultaneous stimuli might induce the CDX2 expression that causes goblet cell metaplasia. BMP4 seems to play an important role in the progress of Barrett's oesophagus. Further investigations are required to elucidate the molecular pathogenesis of Barrett's oesophagus and to develop novel molecular therapeutic strategies aimed at preventing or reversing this pre-malignant condition.

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Conflict of interest statement 

None declared.

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Acknowledgement 

The authors thank Ms. Maki Nomura for assistance of laboratory work.

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PII: S1590-8658(10)00161-1

doi:10.1016/j.dld.2010.04.014

Digestive and Liver Disease
Volume 43, Issue 1 , Pages 54-59, January 2011

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