Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia
Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia
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DOI：
10.1152/ajprenal.00674.2009
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发表时间：
2010-08-01
影响因子：
4.2
通讯作者：
Thomas, Christie P.
中科院分区：
文献类型：
--
作者：
Raikwar, Nandita S.;Vandewalle, Alain;Thomas, Christie P.
Raikwar NS，Vandewalle A，托马斯CP. Nedd 4 -2与occludin相互作用以抑制集合管上皮中紧密连接的形成并增强细胞旁传导。美国肾脏生理学杂志299：F436-F444，2010年。首次发表于2010年5月26日; doi：10.1152/ajprenal.00674.2009。Nedd 4 -2是一种E3泛素连接酶，调节集合管上皮钠通道介导的跨细胞Na+转运。我们研究了Nedd 4 -2对集合管细胞系mpkCCD（c14）细胞连接复合体和细胞旁传导的影响。我们证明，Nedd 4 -2与HEK 293细胞中转染的occludin共免疫沉淀并降低其表达。这种相互作用是通过occludin羧基端保守的PY基序介导的，该PY基序的突变使转染的occludin在HEK 293细胞中的半衰期从6.4小时增加到11.4小时。我们证明，Nedd 4 -2泛素化occludin，这是没有看到当使用Nedd 4 -2的催化失活形式。在mpkCCDc 14细胞中Nedd 4 -2的过表达减少了紧密连接处的闭合蛋白，并在Ca 2+开关测定中短暂增加了细胞旁电导，这与紧密连接形成的延迟一致。相反，siRNA介导的敲除Nedd 4 -2增加了闭合蛋白水平并降低了细胞旁电导。总之，我们证明了Nedd 4 -2在集合管紧密连接组装和细胞旁传导调节中起作用。
Raikwar NS, Vandewalle A, Thomas CP. Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia. Am J Physiol Renal Physiol 299: F436-F444, 2010. First published May 26, 2010; doi:10.1152/ajprenal.00674.2009.-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 mpkCCDc14 cells reduced occludin at the tight junction and transiently increased paracellular conductance in a Ca2+ 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.