Skip to main content

Advertisement

SpringerLink
Log in

Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling

  • Original Article
  • Published:
Journal of Gastrointestinal Surgery Aims and scope

Abstract

Background

The presence of dilated intercellular spaces in the stratified squamous lining of the esophagus is the pathognomonic feature of reflux esophagitis secondary to gastroesophageal reflux disease (GERD). In addition to stomach acid, bile salts are major constituents of gastroesophageal refluxate. The aim of our study was to determine the effect of bile salts cocktail at different pHs on epithelial junctions in an in vitro transwell model of stratified esophageal squamous epithelium.

Discussion

Human telomerase reverse transcriptase (hTERT) immortalized primary esophageal EPC1 cells were grown on polyester transwell surfaces in calcium-enriched media. The cells exhibited gradual stratification into an 11-layered squamous epithelium over 7 days, together with epithelial barrier function as indicated by increased transepithelial electrical resistance (TEER). This stratified epithelium demonstrated well-formed tight junctions, adherens junctions, and desmosomes as visualized by immunofluorescence and electron microscopy. When exposed to short pulses of bile salts at pH 5, but not either condition alone, there was loss of stratification and decrease in TEER, concomitant with disruption of adherens junctions, tight junctions, and desmosomes, leading to the appearance of dilated intercellular spaces. At the cellular level, bile salts at pH 5 activated the Wnt pathway (indicated by increased β-catenin Ser552 phosphorylation).

Conclusion

In conclusion, in our in vitro transwell model bile salts at pH 5, but not bile salts or media at pH 5 alone, modulate Wnt signaling, disrupt different junctional complexes, and cause increased permeability of stratified squamous esophageal epithelium. These changes approximate the appearance of dilated intercellular space similar to that found in GERD patients.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. El-Serag, H.B., Sweet, S., Winchester, C.C., and Dent, J., Update on the epidemiology of gastro-oesophageal reflux disease: a systematic review. Gut, 2013.

  2. DeVault, K.R., Castell, D.O., and American College of, G., Updated guidelines for the diagnosis and treatment of gastroesophageal reflux disease. Am J Gastroenterol, 2005. 100(1): p. 190–200.

    Article  PubMed  Google Scholar 

  3. Souza, R.F., Krishnan, K., and Spechler, S.J., Acid, bile, and CDX: the ABCs of making Barrett’s metaplasia. Am J Physiol Gastrointest Liver Physiol, 2008. 295(2): p. G211-8.

    Article  CAS  PubMed  Google Scholar 

  4. Kauer, W.K., Peters, J.H., DeMeester, T.R., Feussner, H., Ireland, A.P., Stein, H.J., and Siewert, R.J., Composition and concentration of bile acid reflux into the esophagus of patients with gastroesophageal reflux disease. Surgery, 1997. 122(5): p. 874–81.

    Article  CAS  PubMed  Google Scholar 

  5. Orlando, R.C., Powell, D.W., and Carney, C.N., Pathophysiology of acute acid injury in rabbit esophageal epithelium. J Clin Invest, 1981. 68(1): p. 286–93.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Farre, R., van Malenstein, H., De Vos, R., Geboes, K., Depoortere, I., Vanden Berghe, P., Fornari, F., Blondeau, K., Mertens, V., Tack, J., and Sifrim, D., Short exposure of oesophageal mucosa to bile acids, both in acidic and weakly acidic conditions, can impair mucosal integrity and provoke dilated intercellular spaces. Gut, 2008. 57(10): p. 1366–74.

    Article  CAS  PubMed  Google Scholar 

  7. Orlando, R.C., Dilated intercellular spaces and chronic cough as an extra-oesophageal manifestation of gastrooesophageal reflux disease. Pulm Pharmacol Ther, 2011. 24(3): p. 272–5.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Hofmann, A.F. and Mysels, K.J., Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions. J Lipid Res, 1992. 33(5): p. 617–26.

    CAS  PubMed  Google Scholar 

  9. Araki, Y., Katoh, T., Ogawa, A., Bamba, S., Andoh, A., Koyama, S., Fujiyama, Y., and Bamba, T., Bile acid modulates transepithelial permeability via the generation of reactive oxygen species in the Caco-2 cell line. Free Radic Biol Med, 2005. 39(6): p. 769–80.

    Article  CAS  PubMed  Google Scholar 

  10. Yasuda, H., Hirata, S., Inoue, K., Mashima, H., Ohnishi, H., and Yoshiba, M., Involvement of membrane-type bile acid receptor M-BAR/TGR5 in bile acid-induced activation of epidermal growth factor receptor and mitogen-activated protein kinases in gastric carcinoma cells. Biochem Biophys Res Commun, 2007. 354(1): p. 154–9.

    Article  CAS  PubMed  Google Scholar 

  11. Ghatak, S., Reveiller, M., Toia, L., Ivanov, A., Godfrey, T.E., and Peters, J.H., Bile Acid at Low pH Reduces Squamous Differentiation and Activates EGFR Signaling in Esophageal Squamous Cells in 3-D Culture. Journal of Gastrointestinal Surgery, 2013. 17(10): p. 1723–1731.

    Article  PubMed  Google Scholar 

  12. Pai, R., Tarnawski, A.S., and Tran, T., Deoxycholic acid activates beta-catenin signaling pathway and increases colon cell cancer growth and invasiveness. Mol Biol Cell, 2004. 15(5): p. 2156–63.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Harada, H., Nakagawa, H., Oyama, K., Takaoka, M., Andl, C.D., Jacobmeier, B., von Werder, A., Enders, G.H., Opitz, O.G., and Rustgi, A.K., Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation. Mol Cancer Res, 2003. 1(10): p. 729–38.

    CAS  PubMed  Google Scholar 

  14. Deyrieux, A.F. and Wilson, V.G., In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology, 2007. 54(2): p. 77–83.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Liu, T., Zhang, X., So, C.K., Wang, S., Wang, P., Yan, L., Myers, R., Chen, Z., Patterson, A.P., Yang, C.S., and Chen, X., Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells. Carcinogenesis, 2007. 28(2): p. 488–96.

    Article  CAS  PubMed  Google Scholar 

  16. Benson, K., Cramer, S., and Galla, H.J., Impedance-based cell monitoring: barrier properties and beyond. Fluids Barriers CNS, 2013. 10(1): p. 5.

    Article  PubMed Central  PubMed  Google Scholar 

  17. Epstein, F.H., Mittal, R.K., and Balaban, D.H., The esophagogastric junction. New England Journal of Medicine, 1997. 336(13): p. 924–932.

    Article  Google Scholar 

  18. Castell, D.O., Murray, J.A., Tutuian, R., Orlando, R.C., and Arnold, R., Review article: the pathophysiology of gastro-oesophageal reflux disease—oesophageal manifestations. Aliment Pharmacol Ther, 2004. 20 Suppl 9: p. 14–25.

    Article  PubMed  Google Scholar 

  19. de Vries, D.R., Ter Linde, J.J., van Herwaarden, M.A., Schwartz, M.P., Shephard, P., Geng, M.M., Smout, A.J., and Samsom, M., In GERD patients, mucosal repair associated genes are upregulated in non-inflamed oesophageal epithelium. J Cell Mol Med, 2009. 13(5): p. 936–47.

    Article  PubMed Central  PubMed  Google Scholar 

  20. Bjorkman, E.V., Edebo, A., Oltean, M., and Casselbrant, A., Esophageal barrier function and tight junction expression in healthy subjects and patients with gastroesophageal reflux disease: functionality of esophageal mucosa exposed to bile salt and trypsin in vitro. Scand J Gastroenterol, 2013. 48(10): p. 1118–26.

    Article  PubMed  Google Scholar 

  21. Kalabis, J., Wong, G.S., Vega, M.E., Natsuizaka, M., Robertson, E.S., Herlyn, M., Nakagawa, H., and Rustgi, A.K., Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture. Nat Protoc, 2012. 7(2): p. 235–46.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Cavey, M. and Lecuit, T., Molecular bases of cell-cell junctions stability and dynamics. Cold Spring Harb Perspect Biol, 2009. 1(5): p. a002998.

    Article  PubMed Central  PubMed  Google Scholar 

  23. Orlando, R.C., Esophageal mucosal defense mechanisms. GI Motility online, 2006.

  24. Orsulic, S., Huber, O., Aberle, H., Arnold, S., and Kemler, R., E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. Journal of cell science, 1999. 112(8): p. 1237–1245.

    CAS  PubMed  Google Scholar 

  25. Fang, D., Hawke, D., Zheng, Y., Xia, Y., Meisenhelder, J., Nika, H., Mills, G.B., Kobayashi, R., Hunter, T., and Lu, Z., Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem, 2007. 282(15): p. 11221–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Orlando, L.A. and Orlando, R.C., Dilated intercellular spaces as a marker of GERD. Current gastroenterology reports, 2009. 11(3): p. 190–194.

    Article  PubMed  Google Scholar 

  27. Vela, M.F., Craft, B.M., Sharma, N., Freeman, J., and Hazen-Martin, D., Refractory heartburn: comparison of intercellular space diameter in documented GERD vs. functional heartburn. The American journal of gastroenterology, 2010. 106(5): p. 844–850.

    Article  PubMed  Google Scholar 

  28. Orlando, R.C., Mechanisms of reflux-induced epithelial injuries in the esophagus. Am J Med, 2000. 108 Suppl 4a: p. 104S-108S.

    Article  CAS  PubMed  Google Scholar 

  29. DeMeester, T.R., Clinical biology of the Barrett’s metaplasia, dysplasia to carcinoma sequence. Surg Oncol, 2001. 10(3): p. 91–102.

    Article  CAS  PubMed  Google Scholar 

  30. Atanassoff, P.G., Brull, S.J., Weiss, B.M., Landefeld, K., Alon, E., and Rohling, R., The time course of gastric pH changes induced by omeprazole and ranitidine: a 24-hour dose-response study. Anesth Analg, 1995. 80(5): p. 975–9.

    CAS  PubMed  Google Scholar 

  31. Dehn, T.C., Shepherd, H.A., Colin-Jones, D., Kettlewell, M.G., and Carroll, N.J., Double blind comparison of omeprazole (40 mg od) versus cimetidine (400 mg qd) in the treatment of symptomatic erosive reflux oesophagitis, assessed endoscopically, histologically and by 24 h pH monitoring. Gut, 1990. 31(5): p. 509–13.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Hofmann, A. and Hagey, L., Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cellular and Molecular Life Sciences, 2008. 65(16): p. 2461–2483.

    Article  CAS  PubMed  Google Scholar 

  33. Po, H.N. and Senozan, N.M., The Henderson-Hasselbalch equation: Its history and limitations. Journal of Chemical Education, 2001. 78(11): p. 1499–1503.

    Article  CAS  Google Scholar 

  34. Raimondi, F., Santoro, P., Barone, M.V., Pappacoda, S., Barretta, M.L., Nanayakkara, M., Apicella, C., Capasso, L., and Paludetto, R., Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation. Am J Physiol Gastrointest Liver Physiol, 2008. 294(4): p. G906-13.

    Article  CAS  PubMed  Google Scholar 

  35. Hunt, R.H., Armstrong, D., James, C., Chowdhury, S.K., Yuan, Y., Fiorentini, P., Taccoen, A., and Cohen, P., Effect on intragastric pH of a PPI with a prolonged plasma half-life: comparison between tenatoprazole and esomeprazole on the duration of acid suppression in healthy male volunteers. Am J Gastroenterol, 2005. 100(9): p. 1949–56.

    Article  CAS  PubMed  Google Scholar 

  36. Strugala, V., Avis, J., Jolliffe, I.G., Johnstone, L.M., and Dettmar, P.W., The role of an alginate suspension on pepsin and bile acids—key aggressors in the gastric refluxate. Does this have implications for the treatment of gastro-oesophageal reflux disease? J Pharm Pharmacol, 2009. 61(8): p. 1021–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Roy C. Orlando (UNC School of Medicine, NC) for helpful discussions. The study was performed in the Department of Surgery, University of Rochester Medical Center, Rochester, NY. The data in the paper was presented in parts as a poster in Digestive Disease Week, 2013 (Orlando, FL). Parts of the paper also feature in the doctoral thesis of Dr. Sayak Ghatak, Biology, University of Rochester (URL: http://hdl.handle.net/1802/28858).

Author information

Authors and Affiliations

  1. Department of Surgery, University of Rochester Medical Center, Rochester, NY, USA

    Sayak Ghatak, Marie Reveiller & Eileen M. Redmond

  2. Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA

    Liana Toia

  3. Department of Human and Molecular Genetics, Virginia Commonwealth University Medical Center, Richmond, VA, USA

    Andrei I. Ivanov

  4. Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA

    Zhongren Zhou

  5. Department of Surgery, Boston University, Boston, MA, USA

    Tony E. Godfrey

  6. University Hospitals Cleveland, Cleveland, OH, USA

    Jeffrey H. Peters

Authors
  1. Sayak Ghatak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marie Reveiller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liana Toia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrei I. Ivanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhongren Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eileen M. Redmond
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tony E. Godfrey
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeffrey H. Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeffrey H. Peters.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ghatak, S., Reveiller, M., Toia, L. et al. Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling. J Gastrointest Surg 20, 500–509 (2016). https://doi.org/10.1007/s11605-015-3062-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11605-015-3062-2

Keywords

Search

Navigation