Abstract
Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.
Highlights
After transfection of MDCK C7 and HT-29/B6 cells with CRISPR/Cas9, HDR plasmids and sgRNA targeting angulin-1, puromycin-resistant cell clones were screened for angulin1 knockout by Western blotting
It turned out that angulin-1 is involved in water permeability only in the tight epithelial cell line, while both angulin-1 and tricellulin have no effect on water permeability in an intermediatetight epithelium
It can be concluded that the observed clonal variation of the claudins and tricellulin in sum is balanced and provides a constant barrier function of the bicellular tight junctions (bTJs) and tight junctions (tTJs). These results suggested that the effects of angulin-1 and tricellulin relocalization from tTJs to bTJs on epithelial barrier function are controversial [39]
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Adequate transport of solutes and water across epithelial barriers is indispensable for maintaining normal physiological homeostasis in all animals [1,2]. Fluid is moved either across the plasma membranes of the cells that comprise the epithelial layer (transcellular transport) or between these cells (paracellular transport). The discovery of aquaporin water channels provided a first molecular basis for transcellular water movement [3]
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