Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

S-Nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface.

S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.

Novel s-nitrosothiols have potential therapeutic uses for cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene that encodes the CF transmembrane conductance regulatory (CFTR) protein. The majority of wild-type CFTR and virtually all mutant ΔF508 CFTR are degraded before reaching the cell surface. Certain agents and conditions that increase expression and maturation of CFTR enable the protein to function at the cell surface. We and several research groups have reported that S-nitrosoglutathione (GSNO), a class of endogenous S-nitrosothiols, increases the maturation and function of CFTR in human airway epithelial cells. S-nitrosothiols (SNOs) are endogenous molecules with several cell signaling effects and potential relevance to human lung disease. SNOs are normally present in the human airway and have beneficial effects on lung function. Biochemical evidence suggests that SNOs act on post-translational protein modifications through mechanisms involving S-nitrosylation reactions. S-nitrosylation reactions are increasingly recognized to represent metabolically regulated cell signaling processes. Airway epithelial S-nitrosylation signaling disorders have been observed in a range of diseases, including CF. SNO levels are low in CF patients and normal physiological concentrations are effective in increasing CFTR maturation. The mechanisms by which SNOs improve CFTR expression appear to be novel. However, the precise mechanisms by which SNOs exert their beneficial effects are poorly understood. In the near future, we expect to identify the novel mechanisms by which SNO augments CFTR maturation. This information will be critical for optimizing the design and dosing of SNOs that might be used as CFTR corrector therapies in clinical trials.