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Disruption of Epithelial Tight Junctions by Yeast Enhances the Paracellular Delivery of a Model Protein

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Abstract

Purpose

The aim of this study was to investigate the effect of heat-killed yeast cells on the integrity of epithelial tight junctions in vitro.

Methods

Changes in barrier potential of Caco-2 cell monolayers were assessed by transepithelial electrical resistance (TEER) measurements and by an increasing permeability to a marker protein, horse–radish peroxidase (HRP). Visualisation of tight junction disruption was carried out directly through electron microscopy and indirectly through fluorescence confocal microscopy and immunoblotting of the tight junction-associated proteins zonula occludens ZO-1, occludin and actin.

Results

Yeast cells opened tight junctions in a reversible dose- and time-dependent manner, as shown by a decrease in TEER and an increase in HRP permeability. These changes to barrier potential were shown not to be due to cytotoxic effects but due to modulation of the tight junctions. ZO-1, actin and occludin proteins were demonstrated to be involved in yeast-induced tight junction opening through the use of confocal microscopy and western blotting. Electron microscopy confirmed a direct opening of tight junctions after application of yeast.

Conclusion

Yeast modulated epithelial tight junctions in a reversible manner by contraction of the actin cytoskeleton and shift of ZO-1 and occludin tight junction proteins from the membrane to cytoskeletal areas of the cell.

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Abbreviations

DMEM:

dulbecco's modified eagle medium

ECL:

enhanced chemiluminescence reagent

EDTA:

disodium eth ylenediaminetetraacetate

FCS:

foetal calf serum

FITC:

fluorescein isothiocyanate

HRP:

horse–radish peroxidase

Papp :

apparent permeability coefficient

PBS:

phosphate buffered saline

PKC:

protein kinase C

SEM:

scanning electron microscopy

S.E.M.:

standard error of the mean

TEER:

trans-epithelial electrical resistance

TEM:

transmission electron microscopy

ZO-1:

zonula occludens-1

References

  1. J. Miyoshi and Y. Takai. Molecular perspective on tight-junction assembly and epithelial polarity. Adv. Drug Deliv. Rev. 57:815–855 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. P. D. Ward, T. K. Tippin, and D. R. Thakker. Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm. Sci. Technol. Today 3:346–358 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. J. M. Anderson and C. M. Van Itallie. Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol. 269:G467–G475 (1995).

    CAS  PubMed  Google Scholar 

  4. M. Furuse, T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, S. Tsukita, and S. Tsukita. Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol. 123:1777–1788 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. M. Furuse, M. Itoh, T. Hirase, A. Nagasuchi, S. Yonemura, and S. Tsukita. Direct association of occludin with ZO-1 and its possible involvement in the localisation of occludin at tight junction. J. Cell Biol. 127:1617–1626 (1994).

    Article  CAS  PubMed  Google Scholar 

  6. A. S. Fanning, B. J. Jameson, L. A. Jesaitis, and J. M. Anderson. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J. Biol. Chem. 273:29745–29753 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. J. L. Madara, J. Stafford, D. Barenberg, and S. Carlson. Functional coupling of tight junctions and microfilaments in T84 monolayers. Am. J. Physiol. 254:G416–G423 (1988).

    CAS  PubMed  Google Scholar 

  8. N. Hirokawa and L. G. Tilney. Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear. J. Cell Biol. 95:249–261 (1982).

    Article  CAS  PubMed  Google Scholar 

  9. L. A. Lapierre. The molecular structure of the tight junction. Adv. Drug Deliv. Rev. 41:255–264 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. G. Borchard, H. L. Luessen, A. G. De Boer, J. C. Verhoef, C.-M. Lehr, and H. E. Junginger. The potential of mucoadhesive polymers in enhancing the intestinal peptide drug absorption. III: effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro. J. Control. Release 39:131–138 (1996).

    Article  CAS  Google Scholar 

  11. T. Lindmark, N. Schipper, L. Lazorova, A. G. De Boer, and P. Arturrson. Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes. J. Drug Target. 5:215–223 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. C. M. Meaney and C. M. O'Driscoll. A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the Caco-2 cell culture model. Int. J. Pharm. 207:21–30 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. F. A. Dorkoosh, D. Setyaningsih, G. Borchard, M. Rafiee-Tehrani, J. C. Verhoef, and H. E. Junginger. Effects of superporous hydrogels on paracellular drug permeability and cytotoxicity studies in Caco-2 cell monolayers. Int. J. Pharm. 241:35–45 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. E. L. LeCluyse, S. C. Sutton, and J. A. Fix. In vitro effects of long-chain acylcarnitines on the permeability, transepithelial electrical resistance and morphology of rat colonic mucosa. J. Pharmacol. Exp. Ther. 265:955–962 (1993).

    CAS  PubMed  Google Scholar 

  15. T. Kasai, T. Eguchi, N. Ishiwaki, J. Kaneshige, T. Ozeki, and H. Yuasa. Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive. I. AYC as a novel coating material. Int. J. Pharm. 204:53–59 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. H. Yuasa, J. Kaneshige, T. Ozeki, T. Kasai, T. Eguchi, and N. Ishiwaki. Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive. II: effects of curing on the medicine release from AYC-coated tablets. Int. J. Pharm. 209:69–77 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. H. Yuasa, J. Kaneshige, T. Ozeki, T. Kasai, T. Eguchi, and N. Ishiwaki. Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive. III. AYC aqueous coating onto granules and film formation mechanism of AYC. Int. J. Pharm. 237:15–22 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. G. Ranaldi, I. Marigliano, I. Vespignani, G. Perozzi, and Y. Sambuy. The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J. Nutr. Biochem. 13:157–167 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. E. Cabib, R. Roberts, and B. Bowers. Synthesis of the yeast cell wall and its regulation. Ann. Rev. Biochem. 51:763–793 (1982).

    Article  CAS  PubMed  Google Scholar 

  20. P. Arturrson, G. Wilson, I. I. Hassan, C. J. Dix, I. Williamson, R. Shah, and M. Mackay. Transport and permeability properties of human Caco-2 cells: An in vitro model of the intestinal epithelial cell barrier. J. Control. Release 11:25–40 (1990).

    Article  Google Scholar 

  21. I. J. Hidalgo, T. J. Raub, and R. T. Borchardt. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterol. 96:736–749 (1989).

    CAS  Google Scholar 

  22. A. Shaw. Epithelial Cell Culture: A Practical Approach. Oxford University Press, Oxford, 1996.

    Google Scholar 

  23. J. Smith, E. Wood, and M. Dornish. Effect of chitosan on epithelial cell tight junctions. Pharm. Res. 21:43–49 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. U. K. Laemmli. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970).

    Article  CAS  PubMed  Google Scholar 

  25. B. J. Aungst. Intestinal permeation enhancers. J. Pharm. Sci. 89:429–442 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. M. M. Thanou, A. F. Kotzé, T. Scharringhausen, H. L. Lußen, A. G. de Boer, J. C. Verhoef, and H. E. Junginger. Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal Caco-2 cell monolayers. J. Control. Release 64:15–25 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. A. F. Kotzé, H. L. Lueßen, B. J. de Leeuw, A. G. de Boer, J. C. Verhoef, and H. E. Junginger. Comparison of the effect of different chitosan salts and N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2). J. Control. Release 51:35–46 (1998).

    Article  PubMed  Google Scholar 

  28. P. P. Tirumalasetty and J.G. Eley. Evaluation of dodecylmaltoside as a permeability enhancer for insulin using human carcinoma cells. J. Pharm. Sci. 94:246–255 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. X. Sha, G. Yan, Y. Wu, J. Li, and X. Fang. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. Eur. J. Pharm. Sci. 24:477–486 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. V. Dodane, M. A. Khan, and J. R. Merwin. Effect of chitosan on epithelial permeability and structure. Int. J. Pharm. 182:21–32 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. J. M. Smith, M. Dornish, and E. J. Wood. Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption. Biomaterials 26:3269– 3276 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. C. Klingler, U. Kniesel, S. D. Bamforth, H. Wolburg, B. Engelhardt, and W. Risau. Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors. Histochem. Cell Biol. 113:349–361 (2000).

    CAS  PubMed  Google Scholar 

  33. E. B. M. I. Peixoto and C. B. Collares-Buzato. Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains. Cell Struct. Funct. 29:165–178 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. X.-G. Yang, X.-D. Yang, L. Yuan, K. Wang, and D. C. Crans. The permeability and cytotoxicity of insulin-mimetic vanadium compounds. Pharm. Res. 21:1026–1033 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. T. Gershanik, E. Haltner, C. M. Lehr, and S. Benita. Charge-dependent interaction of self-emulsifying oil formulations with Caco-2 cell monolayers: binding, effects on barrier function and cytotoxicity. Int. J. Pharm. 211:29–36 (2000).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Thanks to the BBSRC and Micap plc for funding this project and to Adrian Hick for assisting with preparation of samples for electron microscopy.

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Authors and Affiliations

  1. School of Biochemistry and Microbiology, Garstang Building, University of Leeds, Leeds, LS2 9JT, UK

    Emily Fuller & Edward Wood

  2. Micap plc, Pemberton Business Centre, Richmond Hill, Pemberton, Wigan, WN5 8AA, UK

    Craig Duckham

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  1. Emily Fuller
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Correspondence to Emily Fuller.

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Fuller, E., Duckham, C. & Wood, E. Disruption of Epithelial Tight Junctions by Yeast Enhances the Paracellular Delivery of a Model Protein. Pharm Res 24, 37–47 (2007). https://doi.org/10.1007/s11095-006-9124-0

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  • DOI: https://doi.org/10.1007/s11095-006-9124-0

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