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Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQPs), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish.

The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the “hurricane-like” motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Many organisms survive fluctuating and extreme environmental conditions by manifesting multiple distinct phenotypes during adulthood by means of developmental processes that enable phenotypic plasticity. We report on the discovery of putative plasticity-enabling genes that are involved in transforming the gill of the euryhaline teleost fish, Fundulus heteroclitus, from its freshwater to its seawater gill-type, a process that alters both morphology and function. Gene expression that normally enables osmotic plasticity is inhibited by arsenic. Gene sets defined by antagonistic interactions between arsenic and salinity show reduced transcriptional variation among individual fish, suggesting unusually accurate and precise regulatory control of these genes, consistent with the hypothesis that they participate in a canalized developmental response. We observe that natural selection acts to preserve canalized gene expression in populations of killifish that are most tolerant to abrupt salinity change and that these populations show the least variability in their transcription of genes enabling plasticity of the gill. We found that genes participating in this highly canalized and conserved plasticity-enabling response had significantly fewer and less complex associations with transcriptional regulators than genes that respond only to arsenic or salinity. Collectively these findings, which are drawn from the relationships between environmental challenge, plasticity, and canalization among populations, suggest that the selective processes that facilitate phenotypic plasticity do so by targeting the regulatory networks that gives rise to the response. These findings also provide a generalized, conceptual framework of how genes might interact with the environment and evolve toward the development of plastic traits.

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.

Iron supplementation for hypoferremic anemia could potentiate bacterial growth in the cystic fibrosis (CF) lung, but clinical trials testing this hypothesis are lacking.Twenty-two adults with CF and hypoferremic anemia participated in a randomized, double-blind, placebo-controlled, crossover trial of ferrous sulfate 325mg daily for 6weeks. Iron-related hematologic parameters, anthropometric data, sputum iron, Akron Pulmonary Exacerbation Score (PES), and the sputum microbiome were serially assessed. Fixed-effect models were used to describe how ferrous sulfate affected these variables.Ferrous sulfate increased serum iron by 22.3% and transferrin saturation (TSAT) by 26.8% from baseline (p0.05) but did not affect hemoglobin, sputum iron, Akron PES, and the sputum microbiome.Low-dose ferrous sulfate improved hypoferremia without correcting anemia after 6weeks. We did not observe significant effects on sputum iron, Akron PES, and the sputum microbiome. Although we did not identify untoward health effects of iron supplementation, a larger blinded randomized controlled trial would be needed to fully demonstrate safety.

Background & Aim Background Growing evidence demonstrate that human mesenchymal stromal cells (MSCs) tailor their in vivo anti-inflammatory response depending on the specific inflammatory environment they encounter. Understanding the anti-inflammatory mechanisms of systemic or intratracheal administrated MSCs in a range of lung diseases, including cystic fibrosis (CF), is crucial to refine MSC-based cell therapies. The aim of this study was to determine the effects of the in vivo lung environment in CF patients on MSCs behavior. Methods, Results & Conclusion Methods Clinically utilized MSCs were exposed to bronchoalveolar lavage (BAL) fluids, a surrogate for the in vivo environment, obtained from patients with CF and healthy volunteers, or Gliotoxin. Following exposure, cells and conditioned medium were assessed for cytotoxicity, levels of immunomodulatory cytokines, and the presence of apoptotic markers. BAL-fluid samples were assessed for pro- and anti-inflammatory cytokines, osmolality, protease activity, gliotoxin concentrations, and double-stranded DNA content. Results Following five hours of BAL-fluid exposure, we found that a sub-population of BAL-fluid from CF-patients induced cellular death. Importantly, BAL-fluid from healthy volunteers did not induce any apparent MSCs death. Utilizing an RNAseq approach, we were able to identify the BCL-2 apoptotic pathway, which could be activated by Aspergillus fumigatus (Asp) a common infection seen in CF-patients, and found that only BAL-fluid from Asp+ CF-patients induced MSC death. Furthermore, we found that Gliotoxin, the major toxin produced by Asp, induced MSC death, an effect which was inhibited by addition of dithiothreitol (DTT). Finally, gliotoxin-exposed MSCs were shown to have less therapeutic effect compared to untreated MSCs in an acute lung injured mice model. Conclusions MSCs exposed to BAL-fluids from Aspergillus fumigatus infected CF-patients displayed an increased cellular death, and exposure to the major fungal toxin resulted in a decreased therapeutic capacity of MSCs in vivo.These results highlight the need to understand the effect of the in vivo inflammatory environment on systemic or locally administered MSCs.

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A Δ plcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δ anr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel in airway epithelial cells, plays an important role in maintaining the volume of the airway surface liquid and therefore mucociliary clearance of respiratory pathogens. A recent study has shown that the E3 ubiquitin ligase Neural precursor cells expressed developmentally downregulated (Nedd4–2) ubiquitinates ΔF508-CFTR in pancreatic epithelial cells and that siRNA-mediated silencing of Nedd4–2 increases plasma membrane ΔF508-CFTR. Because the role of Nedd4–2 in regulating wild-type (wt)-CFTR in airway epithelial cells is unknown, studies were conducted to test the hypothesis that Nedd4–2 also ubiquitinates wt-CFTR and regulates its plasma membrane abundance. We found that Nedd4–2 did not affect wt-CFTR Cl− currents in Xenopus oocytes. Likewise, overexpression of Nedd4–2 in human airway epithelial cells did not alter the amount of ubiquitinated wt-CFTR. siRNA knockdown of Nedd4–2 in human airway epithelial cells had no effect on ubiquitination or apical plasma membrane abundance of wt-CFTR. Thus Nedd4–2 does not ubiquitinate and thereby regulate wt-CFTR in human airway epithelial cells.

OBJECTIVES Aztreonam for inhalation solution (AZLI) was recently approved by the FDA for treating cystic fibrosis (CF) patients infected with Pseudomonas aeruginosa. Here we investigated the effect of aztreonam alone or in combination with tobramycin on P. aeruginosa biofilms grown on CF airway epithelial cells. METHODS P. aeruginosa biofilms, produced by laboratory strains or clinical isolates, were formed on confluent CF airway cells before treatment overnight with aztreonam or tobramycin alone or in combination. Alternatively, antibiotics were added 1 h after bacterial inoculation to assess their ability to impair biofilm formation at 5 h. Bacterial cfu remaining after treatment were then determined by plate counting. RESULTS In the absence of antibiotics, all strains developed biofilms that disrupted CF airway epithelial monolayers overnight. Tobramycin reduced the cfu of all strains grown as biofilms. Aztreonam reduced the cfu of some strains by ∼1 log unit without preserving the integrity of cystic fibrosis airway cell monolayers, while decreasing the biofilms of other clinical isolates by ∼4 log units and protecting the monolayers from being compromised. The combination of aztreonam and tobramycin reduced the cfu of two strains by an additional 0.5 and 2 log units, respectively. Of all the mechanisms explored, Psl exopolysaccharide production might explain the variations in biofilm tolerance to aztreonam in some of the strains. CONCLUSIONS Effects of aztreonam on P. aeruginosa biofilms in the in vitro co-culture model are strain-dependent. The simultaneous application of aztreonam and tobramycin may be beneficial for a subset of CF patients by eliminating susceptible P. aeruginosa strains.

Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. population. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.

More effective treatments for acute promyelocytic leukemia (APL) are needed. APL cell treatment with all-trans-retinoic acid (RA) degrades the chimeric, dominant-negative–acting transcription factor promyelocytic leukemia gene (PML)/RARα, which is generated in APL by chromosomal translocation. The E1-like ubiquitin-activating enzyme (UBE1L) associates with interferon-stimulated gene ISG15 that binds and represses PML/RARα protein. Ubiquitin protease UBP43/USP18 removes ISG15 from conjugated proteins. In this study, we explored how RA regulates UBP43 expression and the effects of UBP43 on PML/RARα stability and APL growth, apoptosis, or differentiation. RA treatment induced UBE1L, ISG15, and UBP43 expression in RA-sensitive but not RA-resistant APL cells. Similar in vivo findings were obtained in a transgenic mouse model of transplantable APL, and in the RA response of leukemic cells harvested directly from APL patients. UBP43 knockdown repressed PML/RARα protein levels and inhibited RA-sensitive or RA-resistant cell growth by destabilizing the PML domain of PML/RARα. This inhibitory effect promoted apoptosis but did not affect the RA differentiation response in these APL cells. In contrast, elevation of UBP43 expression stabilized PML/RARα protein and inhibited apoptosis. Taken together, our findings define the ubiquitin protease UBP43 as a novel candidate drug target for APL treatment. Cancer Res; 70(23); 9875–85. ©2010 AACR.

Introduction This cross-sectional study was conducted to assess the relationship between iron levels in the plasma and sputum of cystic fibrosis (CF) patients. Methods Demographic, clinical, and iron-related laboratory data were prospectively obtained from 25 patients with stable clinical features and 14 patients with worsened clinical features since their most recent evaluations. Results Compared to patients with stable clinical features, those who experienced clinical deterioration demonstrated significantly worse lung function and were more frequently malnourished and diabetic. Members of the latter group were also significantly more hypoferremic and had higher sputum iron content than patients with stable clinical features. No significant correlation was found between plasma and sputum iron levels when the groups were analyzed together and separately. Conclusions Sputum iron content does not correlate with iron-related hematologic tests. Hypoferremia is common in CF and correlates with poor lung function and overall health. Pediatr. Pulmonol. 2011; 46:160–165. © 2011 Wiley-Liss, Inc.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl(-) currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Seawater acclimation in killifish, Fundulus heteroclitus , is mediated in part by a rapid (1 h) translocation of CFTR Cl − channels from an intracellular pool to the plasma membrane in gill and increased CFTR-mediated Cl − secretion. This effect is mediated by serum and glucocorticoid-inducible kinase 1 (SGK1), which is stimulated by plasma hypertonicity rather than cortisol. Since arsenic exposure prevents acclimation to seawater by decreasing CFTR protein levels we tested the hypothesis that arsenic (as sodium arsenite) blocks acclimation to seawater by down regulating SGK1 expression. Freshwater adapted killifish were exposed to arsenic (48 h) and transferred to seawater containing arsenic, and SGK and CFTR expression were measured. Arsenic reduced the seawater induced increase in SGK1 mRNA and protein abundance, and reduced both the total amount of CFTR and the amount of CFTR in the plasma membrane. The decrease in membrane CFTR reduced Cl − secretion. Arsenic also increased the amount of ubiquitinated CFTR and its degradation by the lysosome. Thus, we propose a model whereby arsenic reduces the ability of killifish to acclimate to seawater by blocking the seawater induced increase in SGK1, which results in increased ubiquitination and degradation of CFTR.

Gene array studies comparing cystic fibrosis (CF) and non-CF genotypes should reveal factors that explain variability in CF lung disease progression, yielding insights that lead to improved CF care. To date, studies have reached conflicting conclusions, perhaps due to experimental differences and divergent statistical approaches. This review aims: 1) to summarize the findings of four recent gene studies comparing CF and non-CF genotypes, and 2) to reanalyze original data using a recently developed statistical approach, with the aim of identifying genes and paths consistently regulated by the CF genotype. We identified four studies evaluating the effect of the ΔF508-CFTR mutation on human airway epithelial cell gene expression, restricting our investigation to human airway epithelial cell studies whose data were accessible in NCBI's Gene Expression Omnibus or the European Bioinformatic Institute's ArrayExpress. Gene expression patterns showed consistent repression of MHC class I antigen presentation genes in CF human airway epithelia, suggesting a novel mechanistic explanation for poor clearance of viral and bacterial infections by CF patients. We also examined proinflammatory and NF-κB genes, whose induction is widely accepted as a hallmark of the CF genotype, but found little evidence of induction, consistent with a recent review (Machen TE, Am J Physiol Cell Physiol 291: C218–C230, 2006.). In conclusion, our analysis suggests that the CF genotype may impair immune function in airway epithelial cells but may not increase inflammation. Additional studies are required to determine whether MHC class I gene repression in CF reduces antigen presentation at the protein level and whether repression impairs immune function.

The ability of Pseudomonas aeruginosa to form antibiotic-resistant biofilms is thought to account for the inability of current therapies to resolve bacterial infections in the lungs of patients with cystic fibrosis (CF). We recently described a system in which highly antibiotic-resistant P. aeruginosa biofilms grow on human CF airway epithelial cells, and using this system we showed that enhanced iron release from CF cells facilitates the development of such highly antibiotic-resistant biofilms. Given the positive role for iron in biofilm development, we investigated whether the FDA-approved iron chelators deferoxamine and deferasirox would enhance the ability of tobramycin, the primary antibiotic used to treat CF lung infections, to eliminate P. aeruginosa biofilms. The combination of tobramycin with deferoxamine or deferasirox reduced established biofilm biomass by approximately 90% and reduced viable bacteria by 7-log units. Neither tobramycin nor deferoxamine nor deferasirox alone had such a marked effect. The combination of tobramycin and FDA-approved iron chelators also prevented the formation of biofilms on CF airway cells. These data suggest that the combined use of tobramycin and FDA-approved iron chelators may be an effective therapy to treat patients with CF and other lung disease characterized by antibiotic-resistant P. aeruginosa biofilms.

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.

Background: P. aeruginosa chronically colonizes the lung in CF patients and elicits a proinflammatory response. Excessive secretion of IL-6 and IL-8 by CF airway cells in response to P. aeruginosa infection in the CF airway is though to contribute to lung injury. Accordingly, the goal of this study was to test the hypothesis that Corr4a and VRT325, investigational compounds that increase ΔF508-CFTR mediated Cl- secretion in human CF airway cells, reduce the pro-inflammatory response to P. aeruginosa. Methods: IL-6 and IL-8 secretion by polarized CF human airway epithelial cells (CFBE41o-) were measured by multiplex analysis, and ΔF508-CFTR Cl- secretion was measured in Ussing chambers. Airway cells were exposed to P. aeruginosa (PAO1 or PA14) and Corr4a or VRT325. Results: Corr4a and VRT325 increased ΔF508-CFTR Cl- secretion but did not reduce either constitutive IL-6 or IL-8 secretion, or IL-6 and IL-8 secretion stimulated by P. aeruginosa (PA14 or PAO1). Conclusions: Corr4a and VRT325 do not reduce the inflammatory response to P. aeruginosa in human cystic fibrosis airway epithelial cells.

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the c ystic f ibrosis t ransmembrane conductance r egulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S -NEPC [(2S,3S)- trans -3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif -containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif -containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-alpha signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-alpha to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-alpha signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-DeltaTRL), TNF-alpha increased the amount of WtCFTR but not CFTR-DeltaTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-DeltaTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the c ystic f ibrosis t ransmembrane conductance r egulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif , for C FTR i nhibitory f actor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl − ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

We used proximal tubules isolated from the killifish, Fundulus heteroclitus, to examine the effect of environmentally relevant, sublethal levels of arsenic on the function and expression of MRP2, an ABC transporter that transports xenobiotics into urine, including arsenic-glutathione conjugates. Exposure of fish to arsenic as sodium arsenite (4-14 days) increased both MRP2 expression in the apical membrane of proximal tubules and MRP2-mediated transport activity. The level of MRP2 mRNA was not affected, suggesting a posttranslational mechanism of action. Acute exposure of proximal tubules isolated from control fish to 75-375 ppb arsenic decreased mitochondrial function (inner membrane electrical potential). However, in tubules from fish that were preexposed to arsenic (4-14 days), no such effect on mitochondrial function was observed. Thus, chronic in vivo exposure to arsenic induces mechanisms that protect proximal tubules during subsequent arsenic exposure. Upregulation of MRP2 expression and activity is one likely contributing factor.

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

Killifish are euryhaline teleosts that adapt to rapid changes in the salinity of the seawater. It is generally accepted that acclimation to seawater is mediated by cortisol activation of the glucocorticoid receptor (GR), which stimulates CFTR mRNA expression and CFTR-mediated Cl−secretion by the gill. Because there is no direct evidence in killifish that the GR stimulates CFTR gene expression, quantitative PCR studies were conducted to test the hypothesis that cortisol activation of GR upregulates CFTR mRNA expression and that this response is required for acclimation to seawater. Inhibition of the GR by RU-486 prevented killifish from acclimating to increased salinity and blocked the increase in CFTR mRNA. In contrast, inhibition of the mineralocorticoid receptor by spironolactone had no effect on acclimation to seawater. Thus acclimation to increased salinity in killifish requires signaling via the GR and includes an increase in CFTR gene expression. Because arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt GR transcriptional regulation in avian and mammalian systems, studies were also conducted to determine whether arsenic disrupts cortisol-mediated activation of CFTR gene expression in this in vivo fish model and thereby blocks the ability of killifish to acclimate to increased salinity. Arsenic prevented acclimation to seawater and decreased CFTR protein abundance. However, arsenic did not disrupt the GR-induced increase in CFTR mRNA. Thus arsenic blocks acclimation to seawater in killifish by a mechanism that does not disrupt GR-mediated induction of CFTR gene expression.

Background: Serum- and glucocorticoid-inducible kinase-1 (SGK1) increases CFTR Cl currents in Xenopus oocytes by an unknown mechanism. Because SGK increases the plasma membrane expression of other ion channels, the goal of this paper was to test the hypothesis that SGK1 stimulates CFTR Cl currents by increasing the number of CFTR Cl channels in the plasma membrane. Methods: CFTR Cl currents were measured in Xenopus oocytes by the two-electrode voltage clamp technique, and CFTR in the plasma membrane was determined by laser scanning confocal microscopy. Results: wt-SGK1 stimulated CFTR Cl currents by 42% and increased the amount of CFTR in the plasma membrane by 35%. A kinase-dead SGK mutant (K127N) had a dominant-negative effect on CFTR, reducing CFTR Cl currents by 38%. In addition, deletion of the C-terminal PDZ-interacting motif (SGK1-ΔSFL) increased CFTR Cl currents by 108%. Thus, SGK1-ΔSFL was more effective than wt-SGK1 in stimulating CFTR Cl currents. Neither wt-SGK nor the K127N mutant had any effect on Cl currents in oocytes when expressed alone in the absence of CFTR. Conclusion: SGK1 stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. Moreover, the effect of SGK may be mediated by protein-protein interactions involving the PDZ interacting motif.

The fatal genetic disease Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, ΔF508-CFTR, negatively impacts the processing, gating, and stability of CFTR. Existing combination therapies elicit only modest benefit in ΔF508-CFTR patients. Potentiators and correctors address the gating and processing defects of ΔF508-CFTR, respectively, but do not specifically target its stability. Thus, by targeting additional aspects of the mutant CFTR life cycle we may significantly improve CF patient prognosis. Here, we present evidence for a new class of stabilizers that target CFTR half-life. The adaptor protein Disabled-2 (Dab2), is an essential mediator of CFTR endocytosis. The Dab2 Dab homology (DH) domain is required for Dab2 to co-assemble in complex with CFTR, and our lab has shown that DH peptide inhibitors enhance ΔF508-CFTR function alone and even more so when combined with correctors and potentiators. We have now used an engineered ...

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting., IMPORTANCE The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host-virus interplay but has also led to a major advancement in genetic engineering. Recently, increasing evidence suggested that bacteria can co-opt the CRISPR system for functions besides adaptive immunity to phage infection. This study examined one such alternative function, and this report describes the mechanism of type 1-F CRISPR-dependent loss of the biofilm and swarming in the medically relevant opportunistic pathogen Pseudomonas aeruginosa. Since both biofilm formation and swarming motility are important in the virulence of P. aeruginosa, a full understanding of how the CRISPR system can regulate such group behaviors is fundamental to developing new therapeutics.

Cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-)-selective ion channel, is a prototypic member of the ATP-binding cassette transporter superfamily that is expressed in several organs. In these organs, CFTR assembles into large, dynamic macromolecular complexes that contain signalling molecules, kinases, transport proteins, PDZ-domain-containing proteins, myosin motors, Rab GTPases, and SNAREs. Understanding how these complexes regulate the intracellular trafficking and activity of CFTR provides a unique insight into the aetiology of cystic fibrosis and other diseases.

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), ΔF508, leads to the absence of CFTR Cl−channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl−channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl−secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl−secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and ΔF508-CFTR Cl−channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of ΔF508-CFTR.

Background P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.

Transforming growth factor beta (TGFbeta) is a multifunctional cytokine with effects on many cell types. We recently showed that in addition to epithelial barrier enhancing properties, TGFbeta causes diminished cAMP-driven chloride secretion in colonic epithelia, in a manner that is p38 MAPK-dependent. In this study, we sought to further delineate the mechanism behind TGFbeta diminution of chloride secretion. Using colonic and kidney epithelial cell lines, we found that exposure to TGFbeta causes dramatic changes in the expression and localization of the apical membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). In TGFbeta-treated colonic epithelia (T84 and HT-29), CFTR mRNA was significantly reduced 2-24 h post-cytokine exposure. At a time consistent with decreased colonic epithelial secretory responses (16 h), TGFbeta treatment caused diminished intracellular CFTR protein expression (confocal microscopy) and reduced channel expression in the apical membrane during stimulated chloride secretion (biotinylation assay). In comparison, polarized kidney epithelia (MDCK) treated with TGFbeta displayed similarly reduced secretory responses to cAMP stimulating agents; however, a perinuclear accumulation of CFTR was observed, contrasting the diffuse cytoplasmic CFTR expression of control cells. Our data indicate that TGFbeta has profound effects on the expression and subcellular localization of an important channel involved in cAMP-driven chloride secretion, and thus suggest TGFbeta represents a key regulator of fluid movement.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to approximately 90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 +/- 0.09 x 10(-10) cm2/s at 37 degrees C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-DeltaTRL and GFP-CFTR-DeltaTRA) were also mobile with D significantly increased by approximately 60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Delta TRL, and GFP-CFTR-DeltaTRA had similar mobilities (D approximately 12 x 10(-10) cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- DeltaTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 +/- 0.1 x 10(-7) cm2/s. EBP50 diffusion increased by approximately 2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.

Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.

Objectives Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. Methods P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. Results ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. Conclusions Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI(®) (tobramycin) or Cayston(®) (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa.

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al.

The ΔF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and “chemical chaperones” allow ΔF508-CFTR to traffic to the plasma membrane and increase Cl−permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce ΔF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing ΔF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl−secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl−current across the apical plasma membrane. Thus butyrate increased the amount of functional ΔF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl−secretion because of inhibitory effects on Cl−uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on ΔF508-CFTR will allow investigation of ΔF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, DeltaF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, DeltaF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 microM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased DeltaF508-CFTR cell surface expression and DeltaF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370–1394 and 1404–1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

Cystic fibrosis is caused by mutations in the CFTR gene. The most common of these mutations, DeltaF508, results in a protein that is not trafficked to the apical plasma membrane but instead is retained and degraded in the endoplasmic reticulum (ER) by the 26S proteosome. However, this protein is functional upon plasma membrane expression. It has been theoretically estimated that even a modest ( approximately 10%) increase in CFTR-associated chloride conductance can be beneficial in a clinical setting. Thus, understanding basic CFTR biogenesis is important, and identification of prototypical compounds that can increase CFTR expression and trafficking is potentially useful in the development of novel therapeutic strategies to treat cystic fibrosis. We report that mitomycin C (MMC) elicits such a response by increasing CFTR mRNA and protein expression in T-84 and HT-29 cells at very low, non-cytotoxic, pharmacologically relevant concentrations (0.1 microM) leading to enhanced chloride secretion. Thus, MMC may be a useful compound for understanding CFTR regulation and biogenesis.

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.