The Role Of Bone Morphogenetic Protein 4 In The Pathogenesis Of Barrett Esophagus

Background:Barrett esophagus (BE) is an acquired condition in which the normal esophageal squamous epithelium is replaced by columnar epithelium. Most adenocarcinomas of the esophagus develop on a background of BE, which in turn is caused by chronic gastroesophageal reflux disease (GERD). At present acid and bile salts are known important factors that act sinergically to create an environment favoring the development of metaplasic epithelium in patients probably genetically predisposed. Although both of them can directly injury esophageal epithelium and induce BE, the molecular pathogenesis is poorly understood. It is suggested that bone morphogenetic protein 4 (BMP4) ,which is belong to TGF-βsuperfarmily, plays a role in the transformation of normal esophageal squamous cells into columnar cells. BMP4, a factor determines cells fate, is found up-regulated in BE and esophagitis mucosa when compared with normal squamous or non–goblet cell–containing cardiac epithelium and induce the change of cytokeratin expression in esophageal squamous cells. BMP4 was not only shown to play a role in bone formation, but also found to be essential during embryonic development and differentiation of stem cells. So far the mechanism and significance of up-regulation of BMP4 expression in BE is unclear. The aim of the present study was to investigate the role of the BMP pathway in the transformation of normal esophageal squamous cells into columnar cells induced by acid and bile salts.Methods:1. We created a new method to culture esophageal epithelial cells (EECs) in vitro by obtaining biopsy specimens during routine endoscopies, using TrypLETM Express to take the place of trypsin and culturing cells in complete K-SFM medium.2. By the methods of RT-PCR, quantitative real time RT-PCR and Western blot analysis the effects of acid and bile salts on BMP4 mRNA and protein expression in normal EECs were evaluated.3. By the methods of cell counting and Am-blue detection the proliferation of normal EECs after recombinant human BMP4(rhBMP4) incubation was analyzed.4. By the methods of RT-PCR, quantitative real time RT-PCR and Western blot analysis the levels of Villin and CDX2 expression before and after rhBMP4 incubation were detected, by which we could explore the significance of BMP4 in the metaplasia from squamous cells to columnar cells.5. By Western blot analysis the effects of rhBMP4 on activated level of Smad1 and protein expression of ID2 were evaluated, by which we study the role of BMP pathway in intestinal metaplasia induced by BMP4.RESULTS:1. It was convenient to obtain enough living cells by obtaining biopsy specimens during routine endoscopies. TrypLETM Express had higher purity and lower cellular damage compared with trypase. Cells resided at basal zone of esophageal epithelium predominated in our cultured squamous cells and there are no contamination of fibroblasts. After long-term serial subcultivation cells kept good morphous and reproductive activity.2. BMP4 mRNA and protein were not detected in normal EECs. Acid dose-dependently and time-dependently promote BMP4 mRNA and protein expression. The effect of acid at pH4.0 is significantly higher than the control group (p<0.05). Mixture of conjugated bile salts at pH7.2 lightly stimulated the expression of BMP4 and the effect largely increased when pH value is 4.0.3. rhBMP4 dose-dependently inhibited EECs proliferation and activity, which suggested that BMP4 promote apoptosis in the development of BE.4. rhBMP4 dose-dependently up-regulated the expression of Villin and CDX2 which was negative in EECs.5. Villin mRNA and protein expression was absent before acid exposure in normal EECs. While chronic exposure to acid at pH4.0 highly up-regulated villin expression, which was effectively inhibited by Noggin, a specific antagonist of BMP4. In contrast, villin expression was not observed after temporary exposure to acid at pH4.0.6. rhBMP4 fastly activated Smad1 and time-dependently up-regulated protein expression of ID2.The level of P-Smad1 returned to base line at 10min after incubation with rhBMP4 in EECs.Conclusion:1. The method we used to culture esophagus squamous cells was good at overcoming some shortcomings such as limitation of cells resource, predisposed contamination of fibroblast and difficulty of passage when compared with traditional method.2. Gastric acid dose-dependently and time-dependently up-regulated genetic transcription and protein synthesis of BMP4. The effect of bile salts alone was weak, but largely enhanced when combined with gastric acid. It suggested that gastric acid is the primary factor to promote BMP4 expression or induce BE meanwhile bile salts play a synergistic role.3. BMP4 can inhibit cell proliferation and up-regulate villin and CDX2 expression in normal EECs. The up-regulation of BMP4 maybe the keypoint in intestinal metaplasia of esophageal epithelium.4. BMP4 can effectively activate Smad1 and promote expression of ID2 protein in EECs, which suggest BMP pathway plays a role in the development of BE.