The Ubiquitin Ligase Nedd4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon
The Ubiquitin Ligase Nedd4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon
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DOI：
10.1074/jbc.m116.770065
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发表时间：
2017-02-24
影响因子：
4.8
通讯作者：
Rotin, Daniela
中科院分区：
文献类型：
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作者：
Jiang, Chong;Kawabe, Hiroshi;Rotin, Daniela
泛素连接酶Nedd4-like （Nedd4L，或Nedd4-2）结合并调节肾、肺和其他组织中盐吸收上皮上皮Na+通道（ENaC）的稳定性。它在远端结肠中的作用尚不清楚，远端结肠也吸收盐和液体并表达ENaC。采用条件敲除法敲除小鼠肠上皮内的Nedd4L(Nedd4L(f/f)；我们在这里表明，相对于对照组，Nedd4L耗损导致小鼠远端结肠组织中更高的稳态短路电流（Isc）。这种较高的Isc被添加顶端阿米洛利部分降低，基底外侧布美他尼以及基底外侧Cl-的消耗强烈降低，这表明Na+/K+/2Cl(-) （NKCC1/SLC12A2）共转运体和ENaC是Nedd4L在结肠中的靶标。因此，Nedd4L敲除动物的结肠中NKCC1（和ENaC）蛋白丰度增加，表明Nedd4L通常抑制这些蛋白。然而，我们没有观察到Nedd4L和NKCC1之间的共免疫沉淀，这表明Nedd4L间接抑制了NKCC1的表达。低盐饮食显著提高ENaC mRNA和蛋白的表达量，但没有显著提高ENaC活性。虽然限盐也增加了NKCC1蛋白和mRNA的丰度，但并未导致其活性升高（Isc）。这些结果表明NKCC1是体内nedd4l介导下调的新靶点，它与ENaC一起调节远端结肠的离子和液体运输。
The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4L(f/f);Vil-Cre(ERT2)) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl(-) (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and ENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in and (but not ) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.