Nedd4–2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia

Nedd4–2, a E3 ubiquitin ligase, regulates epithelial sodium channel-mediated transcellular Na + transport in the collecting duct. We investigated the effect of Nedd4–2 on the junctional complex and paracellular conductance in mpkCCD c14 cells, a collecting duct cell line. We demonstrate that Nedd4–2 coimmunoprecipitated with and reduced the expression of transfected occludin in HEK293 cells. This interaction was mediated via a conserved PY motif in the COOH terminus of occludin and mutation of this PY motif increased the half-life of transfected occludin in HEK293 cells from 6.4 to 11.4 h. We demonstrate that Nedd4–2 ubiquitinates occludin, which was not seen when a catalytically inactive form of Nedd4–2 was used. Overexpression of Nedd4–2 in mpkCCD c14 cells reduced occludin at the tight junction and transiently increased paracellular conductance in a Ca 2+ switch assay consistent with a delay in the formation of tight junctions. Conversely, siRNA-mediated knockdown of Nedd4–2 increased occludin levels and reduced paracellular conductance. In summary, we demonstrate that Nedd4–2 plays a role in tight junction assembly and the regulation of paracellular conductance in the collecting duct.