Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) glycoconjugates have a glycosylation phenotype of increased fucosylation and/or decreased sialylation when compared with non-CF. A major increase in fucosyl residues linked alpha 1,3 to antennary GlcNAc was observed when surface membrane glycoproteins of CF airway epithelial cells were compared to those of non-CF airway cells. Importantly, the increase in the fucosyl residues was reversed with transfection of CF cells with wild type CFTR cDNA under conditions which brought about a functional correction of the Cl(-) channel defect in the CF cells. In contrast, examination of fucosyl residues in alpha 1,2 linkage by a specific alpha 1,2 fucosidase showed that cell surface glycoproteins of the non-CF cells had a higher percentage of fucose in alpha 1,2 linkage than the CF cells. Airway epithelial cells in primary culture had a similar reciprocal relationship of alpha 1,2- and alpha 1,3-fucosylation when CF and non-CF surface membrane glycoconjugates were compared. In striking contrast, the enzyme activity and the mRNA of alpha 1,2 fucosyltransferase did not reflect the difference in glycoconjugates observed between the CF and non-CF cells. We hypothesize that mutated CFTR may cause faulty compartmentalization in the Golgi so that the nascent glycoproteins encounter alpha 1,3FucT before either the sialyl- or alpha 1,2 fucosyltransferases. In subsequent compartments, little or no terminal glycosylation can take place since the sialyl- or alpha 1,2 fucosyltransferases are unable to utilize a substrate, which is fucosylated in alpha 1,3 position on antennary GlcNAc. This hypothesis, if proven correct, could account for the CF glycophenotype.     

Terminal glycosylation in cystic fibrosis

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.     

Terminal glycosylation of cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (F508/F508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in 1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fuc1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc1,3/4GlcNAc was detected with a specific 1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.     

Hyperacidification in cystic fibrosis: links with lung disease and new prospects for treatment

A new link between the genetic defect and lung pathology in cystic fibrosis (CF) has been established by the recent discovery of an abnormally acidic pH in the organelles of CF respiratory epithelial cells, along with an increased acidity of the CF airway surface liquid. The defect in cystic fibrosis transmembrane resistance regulator (CFTR) results in hyperacidification of the trans-Golgi network, an organelle responsible for glycosylation, and protein- and membrane-sorting in mammalian cells. Hyperacidification and altered surface glycoconjugates might contribute to mucus thickening, bacterial adhesion and colonization, inflammation, and irreversible tissue damage. The increased acidity of the intracellular organelles and of the lung lining in CF could be linked, and both represent potential therapeutic targets.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Cystic Fibrosis: A Disease of Altered Protein Folding

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, CFTR. Previously we demonstrated that the common F508 mutation in the first nucleotide binding domain (NBD1) alters the ability of the domain to fold into a functional three-dimensional structure, providing a molecular explanation for the observation that the mutant CFTR is retained in the endoplasmic reticulum and does not traffic to the apical membrane of affected epithelial cells. Notably, when conditions are altered to promote folding of the mutant protein, it can assume a functional conformation. Correcting the folding defect may have therapeutic benefit for the treatment of cystic fibrosis. Here we summarize these results and discuss the implications in vitro folding studies have for understanding the pathobiology of CF.     

The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa

Background

Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer.

Results

Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ??F508-CFTR (??F-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ??F-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ??F-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ??F-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ??F-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/??F heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/? heterozygotes had no difference in their responses versus +/+ wild-type mice.

Conclusions

Taken altogether, these data suggest that ??F-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ??F-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.     

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.     

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Background

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods

Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results

We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions

CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.     

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in 1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of 2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.     

Respiratory Syncytial Virus Engineered To Express the Cystic Fibrosis Transmembrane Conductance Regulator Corrects the Bioelectric Phenotype of Human Cystic Fibrosis Airway Epithelium In Vitro

Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.Cystic fibrosis (CF) is an autosomal recessive genetic disease that occurs with an incidence of 1 in every 3,400 live Caucasian births in the United States and is one of the most common fatal hereditary diseases in the world (47). CF is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a low-conductance ATP- and cyclic AMP (cAMP)-dependent chloride ion (Cl⁻) channel. More than 1,500 mutations that can lead to various degrees of CF have been found in CFTR. The most common mutation found in individuals of European descent is a deletion of 3 nucleotides in the CFTR gene resulting in the loss of phenylalanine at position 508 of the CFTR protein (ΔF508). This mutation results in the translation of a protein that folds improperly, causing it to be degraded upon exit from the endoplasmic reticulum. Since 90% of the mortality caused by CF results from lung pathology, restoring functional CFTR to the airways of CF patients remains a goal of gene replacement therapeutics for the disease. In the lung, CFTR is expressed by the respiratory epithelium that lines the lumen of the airways, where it is localized to the apical membrane of ciliated cells and the submucosal gland ductal epithelium (20, 40, 48). CFTR is responsible for the movement of Cl⁻ ions across the apical membranes of the airway epithelium and, in combination with sodium ion (Na⁺) transport, it dictates the volume of airway surface liquid that facilitates mucus transport and mucociliary clearance. Lack of functional CFTR in the cell membrane decreases Cl⁻ ion secretion; a net increase in the intracellular Cl⁻ ion concentration is then followed by increased uptake of sodium (Na⁺) ions by epithelial sodium channels (ENaCs). This additional intracellular ion concentration results in a net increase in water uptake into the cell (68). In patients with CF, the fundamental consequence of CFTR dysfunction in the airway is dehydration of the airway surface liquid (ASL) and an increase in the viscosity of the mucus secretions that coat the respiratory tract. This thickened mucus leads to plugging of the airways, in addition to decreased airway clearance, resulting in an increased susceptibility to both bacterial and viral airway pathogens.Early in vitro experiments using the available recombinant adenoviruses (AdV) and adeno-associated viruses (AAV) showed some efficacy in airway cell transduction (29, 67); however, the human clinical trials were less promising due to the low efficiency of CFTR delivery to the appropriate cells and short-lived CFTR expression, primarily as a consequence of the innate and adaptive immune responses (28, 34, 39, 90). Further studies revealed that CAR, the coxsackievirus and AdV receptor, and heparan sulfate, the AAV receptor, are both expressed on the basolateral surface of the human airway, likely providing another explanation for the poor transduction efficiency of airway cells by these vectors when introduced apically (7, 62, 77, 92). More recently, AAV serotypes that transduce the airway epithelium at a much higher rate have been identified, and additional improvements have been made by mutagenesis, capsid shuffling, and directed evolution (24, 36, 52-54, 78, 89). Lentiviral vectors for the delivery of CFTR to CF patients have also been examined, and improvements have been made, but efficiency and safety concerns persist (33, 41, 57, 72, 76, 85). Here, we suggest a potential viral vector to treat CF that naturally targets the airways.In vitro studies in which CF cells and CFTR-corrected CF cells have been mixed in measured ratios have determined that CFTR expression in 6 to 10% of respiratory cells returns Cl⁻ transport to levels similar to those measured in non-CF epithelial cell cultures (2, 42). However, this low level of correction may not repair some of the other associated defects, such as sodium hyperabsorption and mucus dehydration (40). Similar studies performed by mixing airway epithelial cells from CF and non-CF patients to create mixed well-differentiated human airway epithelial cell (HAE) cultures indicated that if 20% of the cells expressed endogenous levels of CFTR, this correlated with 70% of the Cl⁻ channel response measured in cultures made with 100% non-CF cells (25). More recently, infection of HAE cultures with a recombinant parainfluenza virus type 3 (PIV3) vector engineered to express CFTR was shown to fully correct the Cl⁻ transport defect in HAE cultures. In these studies, CFTR delivery to 25% of the surface airway epithelial cells was required to restore airway surface liquid volume and mucus transport to normal non-CF levels (93). Collectively, these in vitro experiments, in relevant airway cell models, suggest that an effective vector for CFTR delivery would need to target at least 25% of the airway surface epithelial cells.Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that infects the ciliated cells of the airway epithelium of the human respiratory tract (94). Most individuals become infected with RSV during the first and second years of life; however, due to incomplete immunity, individuals can be reinfected by RSV throughout their lifetimes. In most cases, infection results in only mild, self-limited, common cold-like symptoms, although a proportion of primary infections do involve lower respiratory tract disease. Serious illness, which typically involves bronchiolitis or pneumonia, is usually restricted to young infants or the frail elderly. Although RSV infects CF patients at the same frequency that it infects their age-matched siblings, CF patients tend to develop more frequent lower respiratory tract illness. It has been shown that CF patients require more frequent hospitalization due to RSV infection when they are young, but this decreases with age, as it does for healthy children (32, 87). Since RSV can infect the lungs of CF patients, it appears that it can not only navigate through the physical barriers of the normal respiratory tract, but can also make its way through the sticky and mucus-rich environment of the CF lung. In addition, RSV has other features that suggest it might have advantages as a gene therapy vector for the delivery of CFTR to the airways of CF patients. RSV has a tropism for the luminal ciliated cells of the airway, which are a relevant target for CFTR gene therapy (40, 48), and RSV has been shown to lack the overt cytopathology of other respiratory viruses, suggesting that it will not rapidly destroy the cells that it infects (94). RSV also has the ability to reinfect, implying that multiple sequential administrations of an RSV-based vector would be possible.Here, we tested the utility of RSV as a CFTR gene transfer vector. The CFTR gene was inserted into four different sites in the RSV genome to obtain a range of expression levels. The vector was then evaluated for the ability to deliver CFTR to the ciliated cells in an in vitro model of the human airway (HAE). We show that RSV delivered CFTR to ciliated cells and resulted in sufficient transduction efficiency and functional CFTR expression to fully correct the Cl⁻ transport bioelectric defect in primary HAE cultures derived from CF patients. These data support continued efforts to explore the utility of RSV-based vectors as potential gene delivery vectors for the treatment of CF lung disease.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl⁻ secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na⁺ absorption. The mechanistic link between missing CFTR and increased Na⁺ absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na⁺ channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na⁺ absorption.     

Biosynthesis of mucin type <Emphasis Type="Italic">O</Emphasis>-glycans: Lack of correlation between glycosyltransferase and sulfotransferase activities and CFTR expression

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 F508 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 F508CFTR 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.     

Innate immune response in CF airway epithelia: hyperinflammatory?

The lack of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the apical membranes of CF airway epithelial cells abolishes cAMP-stimulated anion transport, and bacteria, eventually including Pseudomonas aeruginosa, bind to and accumulate in the mucus. Flagellin released from P. aeruginosa triggers airway epithelial Toll-like receptor 5 and subsequent NF-B signaling and production and release of proinflammatory cytokines that recruit neutrophils to the infected region. This response has been termed hyperinflammatory because so many neutrophils accumulate; a response that damages CF lung tissue. We first review the contradictory data both for and against the idea that epithelial cells exhibit larger-than-normal proinflammatory signaling in CF compared with non-CF cells and then review proposals that might explain how reduced CFTR function could activate such proinflammatory signaling. It is concluded that apparent exaggerated innate immune response of CF airway epithelial cells may have resulted not from direct effects of CFTR on cellular signaling or inflammatory mediator production but from indirect effects resulting from the absence of CFTRs apical membrane channel function. Thus, loss of Cl^–, HCO₃^–, and glutathione secretion may lead to reduced volume and increased acidification and oxidation of the airway surface liquid. These changes concentrate proinflammatory mediators, reduce mucociliary clearance of bacteria and subsequently activate cellular signaling. Loss of apical CFTR will also hyperpolarize basolateral membrane potentials, potentially leading to increases in cytosolic [Ca²⁺], intracellular Ca²⁺, and NF-B signaling. This hyperinflammatory effect of CF on intracellular Ca²⁺ and NF-B signaling would be most prominently expressed during exposure to both P. aeruginosa and also endocrine, paracrine, or nervous agonists that activate Ca²⁺ signaling in the airway epithelia. Pseudomonas aeruginosa; Toll-like receptor; NF-B; oxidative stress; acidic airway surface liquid; calcium     

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Background

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.

Methods

Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.

Results

Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.

Conclusion

In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

     

Alpha1,3fucosyltransferases in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a glycophenotype of aberrant sialylation and/or fucosylation. The CF glycophenotype is expressed on membrane glycoconjugates of CF airway epithelial cells as increased fucosyl residues in alpha1,3/4 linkage to N-acetyl glucosamine, decreased fucosyl residues in alpha1,2 linkage to galactose and decreased sialic acid. To define the cause of this phenotype, the enzyme activity of alpha1,3fucosyltransferase (FucT) was examined in extracts of CF airway epithelial cells with a variety of low molecular weight substrates. Using Galbeta1,4GlcNAc as substrate, the activity was divided into 66% alpha1,3FucT and 34% alpha1,2FucT. mRNA expression examined with probes to FucTIII, IV, and VII showed that the highest expression of two CF cell lines was for FucTIV. Only one CF cell line expressed mRNA for FucTIII. The non CF airway epithelial cells had significant enzyme activity for alpha1,3FucT and strong mRNA expression for FucTIV. Thus as reported previously for alpha1,2FucT, the biochemical capacity for alpha1,3FucT was present in both the CF and non CF cells and can not be the cause of the CF glycophenotype. These results support the hypothesis that wild type CFTR acts in the Golgi and when mutated as in CF, faulty compartmentalization of terminal glycosyltransferases results, yielding the CF glycophenotype.     

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.     

Chemical rescue of ΔF508-CFTR in C127 epithelial cells reverses aberrant extracellular pH acidification to wild-type alkalization as monitored by microphysiometry

Cystic fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel expressed in apical membranes of wet epithelia. Since CFTR is permeable to HCO₃⁻, and may regulate bicarbonate exchangers, it is not surprising evidence of changes in extracellular pH (pHo) have been found in CF. Previously we have shown that tracking pHo can be used to differentiate cells expressing wild-type CFTR from controls in mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. In this study we characterized forskolin-stimulated extracellular acidification rates in epithelia where chemical correction of mutant ΔF508-CFTR converted an aberrant response in acidification (10%+ increase) to wild-type (25%+ decrease). Thus treatment with corrector (10% glycerol) and the resulting increased expression of ΔF508-CFTR at the surface was detected by microphysiometry as a significant reversal from acidification to alkalization of pHo. These results suggest that CFTR activation as well as correction can be detected by carefully monitoring pHo and support findings in the field that extracellular pH acidification may impact the function of airway surface liquid in CF.