Contribution of Tricellulin to Paracellular Water Permeability in Two Epithelial Cell Lines of Different Tightness

Abstract

Large amounts of water molecules pass through epithelia driven by osmotic gradients along two pathways, paracellular, transcellular, or both. The transcellular pathway comprises aquaporin (AQP) water channels and some carriers like SGLT1. The existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until molecular components of the paracellular water transport, claudin‐2 and claudin‐15, were identified. Recently, an effect of angulin‐2 (ILDR1) on water transport was shown, suggesting that the tricellular tight junction (tTJ) is also involved, but the role of its barrier‐forming protein, tricellulin, was not elucidated so far.For analyzing this, two epithelial cell lines featuring properties of tight epithelium and intermediate‐tight epithelium, Madin‐Darby canine kidney cells clone 7 (MDCK C7) and human colon cells (HT‐29/B6), respectively, were stably transfected with shRNA targeting tricellulin. Standard molecular biological techniques were applied to analyze expression, localization and function of transfected/regulated TJ proteins. Transepithelial water transport was measured in clones with unchanged expression of other transport‐relevant proteins before and after TJ modification using mannitol or 4‐kDa dextran as an osmotic gradient.After shRNA transfection into MDCK C7 cells, two knockdown (KD) clones with lowest expression of tricellulin were chosen. Tricellulin KD downregulated the expression of occludin and claudin‐4 and ‐8 but upregulated that of claudin‐1, thus roughly balancing expression of the tightening proteins. TER was reduced in both tricellulin KD clones; however, the permeability to 4‐kDa FITC‐dextran was increased only in the clone exhibiting the highest reduction of tricellulin. Nonetheless, water fluxes induced by a gradient of mannitol or 4‐kDa dextran were greatly increased in both tricellulin KD clones compared with their respective controls. In the intermediate‐tight epithelial cell line, HT‐29/B6, again two tricellulin KD clones were chosen. Tricellulin KD upregulated the expression of tightening claudins (‐1, ‐3 and ‐8) and angulin‐1, but also upregulated the expression of the claudin‐2 and AQP‐4, both forming water channels. On the other hand, AQP‐3 was downregulated. Comparable to what was found in MDCK C7 cells, tricellulin KD caused TER to decrease and permeability to 4‐kDa FITC‐dextran to increase compared to controls. Remarkably, water fluxes were not significantly different between the tricellulin KD clones and their controls.We conclude that in MDCK C7 cells, a cell line devoid of claudin‐2‐based water channels, tricellulin increases water permeability at reduced expression. In contrast, in HT‐29/B6 cells, tricellulin KD does not significantly alter transepithelial water transport, most probably concealed by the presence of larger transport rates mediated by other proteins. All in all, the contribution of tricellulin to overall water transport becomes significant if no other major transport pathways for water are present.Support or Funding InformationSupported by DFG GRK 2318 A3