Expression of tight junction proteins in low and high resistance epithelial cell lines MDCK-C7 and -C11

Abstract

Epithelial tight junctions consist of the transmembranal proteins occludin and several claudins. It is an unresolved puzzle so far, which of these proteins are responsible for forming the paracellular barrier. Expression of the tight junction proteins occludin and claudins 1, 2, 3, 4, and 5 was studied in tight and leaky clones of Madin-Oarby canine kidney (MDCK) cells. As cell adhesion and the paracellular barrier function of epithelia depend on the extracellular calcium concentration, we studied effects of extracellular Ca 2+ on transepithelial electrical resistance (17) and on the expression of occludin and claudins 1, 2, 3, 4, and 5. Methods: Epithelial cell monolayers of Madin-Darby canine kidney (MDCK) cell clones C7 and Cll were grown on porous polycarbonate filters (n = 17-18 / cell strain) at 37 ° C and the resistance was measured on day 7. Ca ~÷ switch experiments were performed employing a 2 or 4 h low extracellular Ca 2+ (2.5/~M) incubation step. Subsequently, tissues were used for membrane protein preparation. The expression of tight junction proteins was detected by immunoblotting. Results: Monolayers of MDCK-Cll revealed features of a low resistance epithelium (52 + 1 g.c--.......~, whereas MDCK-C7 cells exhibited a relatively high resistance (1056 _+ 74 O.cm2). Low extracellular Ca 2+ decreased ? to less than 50 % of initial/7 in monolayers of both clones, without effect of the inhibitor of protein kinases, H7. Occludin and claudin 1, 2, 3, 4, and 5 were detected in all cell lines under investigation. Expression of occludin and claudin 3 did not differ in C7 and Cll. However, claudins 1 and 2 were stronger expressed in Cll cells, whereas the expression of claudin 4 was pronounced in C7. Low Ca 2+ incubation revealed a decrease of claudin 2 in Cll cells whereas other tight junction proteins remained constant. Conclusion: These observations indicate that tight junction contribution by claudins is correlated to barrier properties of mammalian epithelial cell monolayers. In MDCK cells claudins 1 and 2 are strongly expressed in permeable tight junctions, while claudin 4 is predominantly present in tight junctions of low permeability. These observations suggest that barrier properties of mammalian epithelial cells depend on different combinations of claudin proteins. Furthermore, a decrease of claudin 2 during low Ca 2+ incubation was detected, indicating a differential effect of extracellular Ca 2+ on tight junction protein expression.