Barrier function of epithelia is determined by expression and localization of a specific set of claudins within the tight junction. To determine expression profiles of claudins during cell layer formation, membrane fractions of human colon cell lines HT- 29/B6 and Caco-2, as well as kidney tubule cell subclones MDCK I, II, C7, and C11, were analyzed by Western blotting on days 1 and 7 after seeding, representing subconfluent and confluent stages of the cells. Immunfluorescent stainings of tight junction proteins were analyzed by confocal laser-scanning microscopy. Results: During differentiation of cell monolayers, claudin-1, -3, and -4 were detectable at an early stage in the tight junction of leaky cell lines (MDCK II and MDCK C11), whereas claudin-2 appeared after a delay. In contrast, in cells forming a tight barrier (HT-29/B6, Caco-2), claudin-2 could be only detected in early subconfluent stages but was reduced or even lacking in differentiated confluent stages.

Conclusion: Differentiation of epithelial cell monolayers is accompanied with an initial but preliminary barrier formation. In leaky cells with permanent expression of claudin-2, which determines a selective permeability for cations, this protein is expressed with onward differentiation. In tight epithelial cell lines the claudin-2 expression is reduced or is even lacking in differentiated confluent stages but can be detected in early subconfluent stages.