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Cystic Fibrosis (CF) is due to a folding defect in the CFTR protein. The most common mutation [ΔF508] prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-Ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser422. SGK1[pS422] and C4-CER co-incidently bind PDK1 and permit PDK1 to autophosphorylate at Ser241. Then, PDK1 [pS241] phosphorylates SGK1[pS422] at Thr256 to generate fully activated SGK1 [pS422, pT256]. SGK1[pS422, pT256] then phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. [ΔF508]CFTR is thus free to traffick to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mTOR signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of [ΔF508]CFTR (t1/2 ~ 10 h); enhances cAMP-activated chloride secretion; and suppresses hypersecretion of Interleukin-8 (IL-8). We suggest that therapies for CF directed against the PDK1/SGK1 signaling pathway, such as that provided by C4-CER, is a novel therapeutic strategy for a life-limiting disorder which affects one child, on average, each day. [ABSTRACT FROM AUTHOR]

The most common mutation in cystic fibrosis (CF) is ΔF508, 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 corEectly trafficked ΔF508-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 serumand glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of ΔF508-CFTR. The half-life of ΔF508-CFTR is also dramatically enhanced. Dexamethasone-activated ΔF508-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-typeand ΔF508-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 ΔF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated ΔF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein 11-8, and the high levels of 11-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of 11-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-κB (lκBa). lκBα phosphorylation is a required step in the activation of the NF-κB signaling pathway and the subsequent expression of 11-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing 11-8-dependent lung inflammation in CF. [ABSTRACT FROM AUTHOR]