The most common mutation in cystic fibrosis (CF) is ⌬F508, 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 ⌬F508-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 ⌬F508-CFTR. The half-life of ⌬F508-CFTR is also dramatically enhanced. Dexamethasone-activated ⌬F508-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 ⌬F508-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 ⌬F508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated ⌬F508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.

Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.

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 IL-8, and the high levels of IL-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 IL-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-B (I B␣). I B␣ phosphorylation is a required step in the activation of the NF-B signaling pathway and the subsequent expression of IL-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 IL-8-dependent lung inflammation in CF.

Digitoxin and structurally related cardiac glycoside drugs potently block activation of the TNF-␣͞NF-B signaling pathway. We have hypothesized that the mechanism might be discovered by searching systematically for selective inhibitory action through the entire pathway. We report that the common action of these drugs is to block the TNF-␣-dependent binding of TNF receptor 1 to TNF receptor-associated death domain. This drug action can be observed with native cells, such as HeLa, and reconstituted systems prepared in HEK293 cells. All other antiinflammatory effects of digitoxin on NF-B and c-Jun N-terminal kinase pathways appear to follow from the blockade of this initial upstream signaling event.digitoxin ͉ inflammation

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Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2-DE and/or MS need to be complemented by different kinds of technologies. The well-known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody-centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label-free detection technologies, human genome-scale numbers of analytes, and increases in analytic and bioinformatic speeds.

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Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3′-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.