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Effect of RNA interference of tight junction-related molecules on intercellular barrier function in cultured human keratinocytes

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Abstract

Accumulating evidence shows that tight junctions (TJs) in the granular layer contribute to the epidermal barrier, suggesting that the regulation of TJ assembly in keratinocytes may provide a clue to understanding the role of barrier formation in epidermis. In this study, we investigated the behavior of TJ-related molecules in cultured human keratinocytes during keratinization induced by transfer to high-calcium medium, and the effect of RNA interference of TJ-related molecules on intercellular permeability and morphological features. The expression of TJ-related molecules and transepithelial electrical resistance were increased by transfer to high-calcium medium. In cells under the same conditions, we observed by freeze-fracture electron microscopy that TJ strands developed on the apposing cell membranes. In contrast, the transepithelial electrical resistance was clearly suppressed when the expression of claudin-1 or occludin was blocked by RNA interference. The morphological features of these knock-down cells were the same as those of MOCK cells, except for a marked decrease in the number of TJ strands. Furthermore, claudin-1 suppression inhibited occludin localization at the cell membrane, whereas suppression of occludin did not influence the localization of claudin-1. These results suggest that claudin-1 plays a crucial role in recruiting occludin to TJs, and that occludin is involved in intercellular barrier function.

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Abbreviations

CL1kd:

Claudin-1 knock-down

FITC-conjugated:

Fluorescein isothiocyanate-conjugated

HEK:

Human epidermal keratinocyte

MOCK:

Mock transfected

OCLkd:

Occludin knock-down

PB:

Phosphate buffer

PBS:

Phosphate-buffered saline

siRNA:

Small interfering RNA

TER:

Transepithelial electrical resistance

TJ:

Tight junction

ZO-1:

Zonula occludens-1

References

  1. Anderson JM, Van Itallie CM, Fanning AS (2004) Setting up a selective barrier at the apical junction complex. Curr Opin Cell Biol 16:140–145

    Article  PubMed  CAS  Google Scholar 

  2. Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, Itoh M, Yonemura S, Furuse M, Tsukita S (1996) Interspecies diversity of the occludin sequence cDNA cloning of human, mouse, dog, and rat-kangaroo homologues. J Cell Biol 133:43–47

    Article  PubMed  CAS  Google Scholar 

  3. Bikle DD, Pillai S (1993) Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14:3–19

    Article  PubMed  CAS  Google Scholar 

  4. Brandner JM, Kief S, Grund C, Rendl M, Houdek P, Kuhn C, Tschachler E, Franke WW, Moll I (2002) Organization and formation of the tight junction system in human epidermis and cultured keratinocytes. Eur J Cell Biol 81:253–263

    Article  PubMed  CAS  Google Scholar 

  5. Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227–1237

    Article  PubMed  CAS  Google Scholar 

  6. Eckert RL, Green H (1986) Structure and evolution of the human involucrin gene. Cell 46:583–589

    Article  PubMed  CAS  Google Scholar 

  7. Elias PM (1996) The stratum corneum revisited. J Dermatol 23:756–758

    PubMed  CAS  Google Scholar 

  8. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–1550

    Article  PubMed  CAS  Google Scholar 

  9. Furuse M, Furuse K, Sasaki H, Tsukita S (2001) Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin–Darby canine kidney I cells. J Cell Biol 153:263–272

    Article  PubMed  CAS  Google Scholar 

  10. Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, Noda T, Kubo A, Tsukita S (2002) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111

    Article  PubMed  CAS  Google Scholar 

  11. Helfrich I, Schmitz A, Zigrino P, Michels C, Haase I, Le Bivic A, Leitges M, Niessen CM (2007) Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation. J Invest Dermatol 127:782–791

    Article  PubMed  CAS  Google Scholar 

  12. Hennings H, Holbrook KA (1983) Calcium regulation of cell–cell contact and differentiation of epidermal cells in culture. An ultrastructural study. Exp Cell Res 143:127–142

    Article  PubMed  CAS  Google Scholar 

  13. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–254

    Article  PubMed  CAS  Google Scholar 

  14. Kitajima Y, Eguchi K, Ohno T, Mori S, Yaoita H (1983) Tight junctions of human keratinocytes in primary culture: a freeze-fracture study. J Ultrastruct Res 82:309–313

    Article  PubMed  CAS  Google Scholar 

  15. Langbein L, Grund C, Kuhn C, Praetzel S, Kartenbeck J, Brandner JM, Moll I, Franke WW (2002) Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom. Eur J Cell Biol 81:419–435

    Article  PubMed  CAS  Google Scholar 

  16. Langbein L, Page UF, Grund C, Kuhn C, Praetzel S, Moll I, Moll R, Franke WW (2003) Tight junction-related structures in the absence of a lumen: occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas. Eur J Cell Biol 82:385–400

    Article  PubMed  CAS  Google Scholar 

  17. Leyvraz C, Charles RP, Rubera I, Guitard M, Rotman S, Breiden B, Sandhoff K, Hummler E (2005) The epidermal barrier function is dependent on the serine protease CAP1/Prss8. J Cell Biol 170:487–496

    Article  PubMed  CAS  Google Scholar 

  18. McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, Schneeberger EE (1996) Occludin is a functional component of the tight junction. J Cell Sci 109:2287–2298

    PubMed  CAS  Google Scholar 

  19. Mehrel T, Hohl D, Rothnagel JA, Longley MA, Bundman D, Cheng C, Lichti U, Bisher ME, Steven AC, Steinert PM, Yuspa H, Roop DR (1990) Identification of a major keratinocyte cell envelope protein, loricrin. Cell 61:1103–1112

    Article  PubMed  CAS  Google Scholar 

  20. Morita K, Furuse M, Fujimoto K, Tsukita S (1999) Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 96:511–516

    Article  PubMed  CAS  Google Scholar 

  21. Nemes Z, Steinert PM (1999) Bricks and mortar of the epidermal barrier. Exp Mol Med 31:5–19

    PubMed  CAS  Google Scholar 

  22. Nusrat A, Brown GT, Tom J, Drake A, Bui TT, Quan C, Mrsny RJ (2005) Multiple protein interactions involving proposed extracellular loop domains of the tight junction protein occludin. Mol Biol Cell 16:1725–1734

    Article  PubMed  CAS  Google Scholar 

  23. Pummi K, Malminen M, Aho H, Karvonen SL, Peltonen J, Peltonen S (2001) Epidermal tight junctions: ZO-1 and occludin are expressed in mature, developing, and affected skin and in vitro differentiating keratinocytes. J Invest Dermatol 117:1050–1058

    Article  PubMed  CAS  Google Scholar 

  24. Read J, Watt FM (1988) A model for in vitro studies of epidermal homeostasis: proliferation and involucrin synthesis by cultured human keratinocytes during recovery after stripping off the suprabasal layers. J Invest Dermatol 90:739–743

    Article  PubMed  CAS  Google Scholar 

  25. Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T, Tsukita S (2000) Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell 11:4131–4142

    PubMed  CAS  Google Scholar 

  26. Sawada N, Murata M, Kikuchi K, Osanai M, Tobioka H, Kojima T, Chiba H (2003) Tight junctions and human diseases. Med Electron Microsc 36:147–156

    Article  PubMed  Google Scholar 

  27. Schneeberger EE, Lynch RD (2004) The tight junction: a multifunctional complex. Am J Physiol Cell Physiol 286:C1213–C1228

    Article  PubMed  CAS  Google Scholar 

  28. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP (1999) Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 285:103–106

    Article  PubMed  CAS  Google Scholar 

  29. Stanley JR, Yuspa SH (1983) Specific epidermal protein markers are modulated during calcium-induced terminal differentiation. J Cell Biol 96:1809–1814

    Article  PubMed  CAS  Google Scholar 

  30. Stevenson BR, Keon BH (1998) The tight junction: morphology to molecules. Annu Rev Cell Dev Biol 14:89–109

    Article  PubMed  CAS  Google Scholar 

  31. Tsukita S, Furuse M (2000) Pores in the wall: claudins constitute tight junction strands containing aqueous pores. J Cell Biol 149:13–16

    Article  PubMed  CAS  Google Scholar 

  32. Tsukita S, Furuse M (1998) Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol 9:268–273

    Article  Google Scholar 

  33. Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2:285–293

    Article  PubMed  CAS  Google Scholar 

  34. Turksen K, Troy TC (2002) Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129:1775–1784

    PubMed  CAS  Google Scholar 

  35. Umeda K, Matsui T, Nakayama M, Furuse K, Sasaki H, Furuse M, Tsukita S (2004) Establishment and characterization of cultured epithelial cells lacking expression of ZO-1. J Biol Chem 279:44785–44794

    Article  PubMed  CAS  Google Scholar 

  36. Van Itallie CM, Anderson JM (1997) Occludin confers adhesiveness when expressed in fibroblasts. J Cell Sci 110:1113–1121

    PubMed  Google Scholar 

  37. Watt FM (1983) Involucrin and other markers of keratinocyte terminal differentiation. J Invest Dermatol 81:100S–103S

    Article  PubMed  CAS  Google Scholar 

  38. Yamamoto T, Kurasawa M, Hattori T, Maeda T, Nakano H, Sasaki H (2008) Relationship between expression of tight junction-related molecules and perturbed epidermal barrier function in UVB-irradiated hairless mice. Arch Dermatol Res 300:61–68

    Article  PubMed  CAS  Google Scholar 

  39. Yoshida Y, Morita K, Mizoguchi A, Ide C, Miyachi Y (2001) Altered expression of occludin and tight junction formation in psoriasis. Arch Dermatol Res 293:239–244

    Article  PubMed  CAS  Google Scholar 

  40. Yuki T, Haratake A, Koshikawa H, Morita K, Miyachi Y, Inoue S (2007) Tight junction proteins in keratinocytes: localization and contribution to barrier function. Exp Dermatol 16:324–330

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Clara Franzini-Armstrong, professor at the Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine and Dr. Yasuo Kitajima, professor and chairman of the Department of Dermatology, Gifu University, for their helpful discussions.

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Authors and Affiliations

  1. Department of Dermatology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto, Tokushima, 770-8503, Japan

    Takuya Yamamoto & Seiji Arase

  2. Pola Chemical Industries Inc., 560 Kashio-cho, Totsuka-ku, Yokohama, 244-0812, Japan

    Takuya Yamamoto, Yuko Saeki, Masumi Kurasawa & Shohei Kuroda

  3. Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan

    Hiroyuki Sasaki

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  1. Takuya Yamamoto
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  2. Yuko Saeki
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  3. Masumi Kurasawa
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Correspondence to Hiroyuki Sasaki.

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Yamamoto, T., Saeki, Y., Kurasawa, M. et al. Effect of RNA interference of tight junction-related molecules on intercellular barrier function in cultured human keratinocytes. Arch Dermatol Res 300, 517–524 (2008). https://doi.org/10.1007/s00403-008-0868-8

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  • DOI: https://doi.org/10.1007/s00403-008-0868-8

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