Caohuy Hung, Jozwik Catherine, Pollard Harvey B
Department of Anatomy, Physiology, and Genetics, Uniformed Services University School of Medicine, Bethesda, Maryland 20814, USA.
J Biol Chem. 2009 Sep 11;284(37):25241-53. doi: 10.1074/jbc.M109.035345. Epub 2009 Jul 17.
The most common mutation in cystic fibrosis (CF) is DeltaF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked DeltaF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of DeltaF508-CFTR. The half-life of DeltaF508-CFTR is also dramatically enhanced. Dexamethasone-activated DeltaF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and DeltaF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects DeltaF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated DeltaF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.
囊性纤维化（CF）最常见的突变是ΔF508，它与突变的囊性纤维化跨膜传导调节因子（CFTR）无法转运至质膜有关。通过一种尚不清楚的机制，正确转运的ΔF508-CFTR的缺失导致顶端质膜上的上皮钠通道（ENaC）过量。已知ENaC的转运受一条涉及糖皮质激素受体、血清和糖皮质激素调节激酶SGK1以及泛素E3连接酶Nedd4-2的信号通路调控。我们在此表明，地塞米松可挽救ΔF508-CFTR的功能表达。ΔF508-CFTR的半衰期也显著延长。地塞米松激活的ΔF508-CFTR挽救作用被糖皮质激素受体拮抗剂RU38486或磷脂酰肌醇3-激酶抑制剂LY294002阻断。免疫共沉淀研究表明，Nedd4-2与野生型和ΔF508-CFTR均结合。这些复合物被地塞米松处理所抑制，同时CFTR的泛素化减少。我们进一步表明，小干扰RNA敲低Nedd4-2也能纠正ΔF508-CFTR的转运。相反，小干扰RNA敲低SGK1则完全阻断地塞米松激活的ΔF508-CFTR挽救作用。这些数据表明，SGK1/Nedd4-2信号通路在CF上皮细胞中调节CFTR和ENaC的转运。
