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The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl− conductance (gCFTR) in the apical membrane is completely deactivated following α-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through α-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10−8 M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

Airway surface hydration depends on the balance between transepithelial Na+ absorption and Cl- secretion. In adult mice, absence of functional cystic fibrosis transmembrane conductance regulator (Cftr) fails to recapitulate human cystic fibrosis (CF) lung disease. In contrast, overexpression of the epithelial Na+ channel β subunit in transgenic mice (βENaC-Tg) produces unregulated Na+ hyperabsorption and results in CF-like airway surface dehydration, mucus obstruction, inflammation, and increased neonatal mortality. To investigate whether the combination of airway Na+ hyperabsorption and absent Cftr-mediated Cl- secretion resulted in more severe lung pathology, we generated doublemutant ΔF508 CF/βENaC-Tg mice. Survival of ΔF508 CF/ βENaC-Tg mice was reduced compared with ΔENaC-Tg or βF508 CF mice. Absence of functional Cftr did not affect endogenous or transgenic ENaC currents but produced reduced basal components of Cl- secretion and tracheal cartilaginous defects in both ΔF508 CF and ΔF508 CF/βENaC-Tg mice. Neonatal ΔF508 CF/βENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with βENaC-Tg littermates. Neonatal ΔF508 CF/βENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of βENaC-Tg littermates. Adult ΔF508 CF/βENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched βENaC-Tg mice. Collectively, these data suggest that the combined abnormalities in Na+absorption and Cl- secretion produce more severe lung disease than either defect alone. Airway cartilage abnormalities, airway cell necrosis, and exaggerated neutrophil infiltration likely interact with defective mucus clearance caused by βENaC overexpression and absent CFTR-mediated Cl- secretion to produce the increased neonatal mortality observed in ΔF508 CF/βENaC-Tg mice. [ABSTRACT FROM AUTHOR]

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of ΔF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of ΔF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR. [Copyright &y& Elsevier]

ΔF508 CFTR can be functionally restored in the plasma membrane by exposure of the cell to lower temperature. However, restored ΔF508 CFTR has a much shorter half-life than normal. We studied whether NHERF1, which binds to the PDZ motif of CFTR, might be a critical mediator in the turnover of ΔF508 CFTR from the cell surface. We used RNAi to reduce the expression of NHERF1 in human airway epithelial cells. Knockdown of NHERF1 reversibly reduces surface expression of WT-CFTR without altering its total expression. As expected, temperature correction increased mature C band ΔF508 CFTR (rΔF508) but unexpectedly allowed immature B band of rΔF508 to traffic to the cell surface. Both surface and total expression of rΔF508 in NHERF1 knockdown cells were reduced and degradation of surface localized rΔF508 was even faster in NHERF1 knockdown cells. Proteasomal and lysosomal inhibitor treatments led to a significant decrease in the accelerated degradation of surface rΔF508 in NHERF1 knockdown cells. These results indicate that NHERF1 plays a role in the turnover of CFTR at the cell surface, and that rΔF508 CFTR at the cell surface remains highly susceptible to degradation. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Curcumin has been reported to correct cystic fibrosis caused by the ΔF508 mutation of the cystic fibrosis transmembrane regulator (CFTR) but its mechanistic action remains unclear. We have recently demonstrated that the ER chaperone calreticulin (CRT) negatively regulates the CFTR cell surface expression and activity. Thus, we aimed at determining whether CRT mediates the effect of curcumin on CFTR. We show here that the treatment with curcumin of Chinese hamster ovary cells suppressed CRT expression and increased wild-type CFTR but did not affect ΔF508 CFTR expression. However, we determined that although curcumin did not augment ΔF508 CFTR expression, it enhanced the functional competence of ΔF508 CFTR induced by 26°C incubation. Knock down of CRT by siRNA at low-temperature had a similar effect. Our findings suggest that the positive effect of curcumin on CFTR expression is mediated through the down-regulation of CRT, a negative regulator of CFTR. [Copyright &y& Elsevier]

Vitamin C (L-ascorbate) is present in the respiratory lining fluid of human lungs, and local deficits occur during oxidative stress. Here we report a unique function of vitamin C on the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-dependent Cl channel that regulates epithelial surface fluid secretion. Vitamin C (100 μM) induced the openings of CFTR Cl channels by increasing its average open probability from 0 to 0.21 ± 0.08, without a detectable increase in intracellular cAMP levels. Exposure of the apical airway surface to vitamin C stimulated the transepithelial Cl secretion to 68% of forskolin-stimulated currents. The average half-maximal stimulatory constant was 36.5 ± 2.9 μM, which corresponds to physiological concentrations. When vitamin C was instilled into the nasal epithelium of human subjects, it effectively activated Cl transport in vivo. In CF epithelia, previous treatment of the underlying trafficking defect with trimethylamine oxide or expression of WT CFTR restored the activation of Cl transport by vitamin C. Sodium dependency and phloretin sensitivity, as well as the expression of transcripts for sodium-dependent vitamin C transporter (SVCT)-1 and SVCT2, support a model in which an apical vitamin C transporter is central for relaying the effect of vitamin C to CFTR. We conclude that cellular vitamin C is a biological regulator of CFTR-mediated Cl secretion in epithelia The pool of vitamin C in the respiratory tract represents a potential nutraceutical and pharmaceutical target for the complementary treatment of sticky airway secretions by enhancing epithelial fluid secretion. [ABSTRACT FROM AUTHOR]

Structural differences have been reported in the glycosylation patterns of cystic fibrosis glycoproteins. Although the gene mutated in cystic fibrosis (CFTR) has been cloned and characterized as a chloride channel, its relationship to the highly viscous mucus and structural glycoprotein and mucin abnormalities in cystic fibrosis still remains to be defined. We have evaluated O-glycan biosynthesis in CHO and BHK cells that express CFTR and ΔF508 CFTR as in vitro models, and utilized the cftr knockout mouse as an in vivo model of CFTR dysfunction. Activities of glycosyltransferases and sulfotransferases synthesizing mucin type O-glycan chains were determined in these models. Differences in transferase activity levels were found between tissues and cell types and during mouse development. No specific patterns of activities were associated with the lack of CFTR or with ΔF508CFTR expression. This suggests that it is not the presence or absence of normal CFTR, or the presence of mutant CFTR alone, but rather cell specific additional factors or pathophysiological consequences that determine the changes in mucin glycosylation in cystic fibrosis. [ABSTRACT FROM AUTHOR]

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a 'tight epithelium' in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, ΔF508 homozygote, cell line that has tight junctions pre-crisis. The cells (ΣCFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl transport as measured by efflux of radioactive Cl, short circuit current (I), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K- and Ca-dependent Cl transport. Measurement ofCl efflux following stimulation with A23187 and ionomycin indicate a Ca-dependent Cl transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26° C for 48 h there is no indication of cAMP-dependent Cl as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells. Gene complementation studies with an episomal vector containing wild-type CF transmembrane conductance regulator cDNA showed correction of the cAMP-dependent Cl transport defect. This cell line contributes unique phenotypic features to the store of transformed CF epithelial cells already available. [ABSTRACT FROM AUTHOR]