Contribution of the tricellular tight junction proteins, tricellulin and angulin-1, to paracellular water permeability

Abstract

In epithelia, large amounts of water pass by transcellular and paracellular pathways, driven by the osmotic gradient built up by solutes’ movement. The transcellular pathway has been molecularly characterized by the discovery of aquaporin membrane channels. Unlike this, the existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until two molecular components of paracellular water transport, claudin-2, and claudin-15, were identified. A main protein of the tricellular TJ (tTJ), tricellulin, was shown to be downregulated in ulcerative colitis leading to increased permeability to macromolecules. In addition to tricellulin, the family of angulin proteins is known to be able to recruit tricellulin to the tTJ. Recently, Gong and colleagues found that angulin-2 knockout increases water transport on isolated mouse kidney tubules, and its overexpression reduces the water transport in MDCK II cells. Whether or not tricellulin regulates water transport or angulin-1 also mediates a direct effect on water permeability independent of tricellulin, or act indirectly via tricellulin regulation, is unknown yet. To answer our research question, two epithelial cell lines featuring properties of the tight and intermediate-tight epithelium, MDCK C7 and HT-29/B6, respectively, were stably transfected with shRNA targeting tricellulin or sgRNA along with CRISPR/Cas9 targeting angulin-1, proteins of the tTJ essential for the barrier against passage of solutes up to 10 kDa. Interestingly, tricellulin knockdown and angulin-1 knockout reduced the transepithelial resistance (TER) in both cell lines and increased 4-kDa FITC-dextran permeability, especially in HT-29/B6 cell line. In addition, the expression and location of different TJ proteins in control cells and in knockdown or knockout clones were investigated. In MDCK C7 cells, tricellulin knockdown and angulin-1 knockout downregulated the expression of some tight junction proteins, while on HT-29/B6 cells, the opposite effect was observed; some TJ proteins were upregulated. Finally, water flux was induced by osmotic gradients using mannitol, 4-kDa, and 40-kDa dextran or albumin and measured in both cell lines. In MDCK C7 cells, tricellulin knockdown and angulin-1 knockout increased the water flux compared to that of vector controls, indicating a direct role of tricellulin in regulating water permeability in a tight epithelial cell line; nevertheless, in HT-29/B6 cells, water flux was unchanged between the control and the tricellulin knockdown or angulin-1 knockout clones. We conclude that tricellulin and angulin-1, the latter acting indirectly via tricellulin displacement, increase water permeability at reduced expression only in MDCK C7 cells, i.e., in the tight epithelium.