Retinoic acid-induced glandular differentiation of the oesophagus

Abstract

Background: Retinoic acid (RA) is a powerful differentiation agent. Barrett’s oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett’s oesophagus. Methods: RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett’s oesophagus cell lines with or without LCA. SE and Barrett’s oesophagus biopsy specimens were exposed to 100 μM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. Results: RA biosynthesis was increased in Barrett’s oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett’s oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. Conclusion: RA activity is increased in Barrett’s oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.