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.     

Cigarette Smoke-induced Ca2+ Release Leads to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Dysfunction

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca²⁺ that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca²⁺, but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca²⁺ release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca²⁺ and CFTR internalization. We hypothesize that CS-induced Ca²⁺ release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment.     

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism.     

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.     

Extracellular pH and lung infections in cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na⁺/K⁺ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.     

Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl(-) secretion and increased airway Na(+) absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na(+) transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na(+) channels (ENaC). Here we show that increased airway Na(+) absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na(+) absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease.     

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.     

CFTR mutations and host susceptibility to Pseudomonas aeruginosa lung infection

The susceptibility of cystic fibrosis patients to bacterial pathogens is associated with deficient airway antimicrobial peptide activity, and airway-surface-liquid dehydration with decreased transport velocity and hypersecretion of mucus. Susceptibility to Pseudomonas aeruginosa infection has been linked to the role of the cystic fibrosis transmembrane conductance regulator protein as a receptor for P. aeruginosa. Binding of P. aeruginosa coordinates lung clearance as part of innate immunity. The function of CFTR in innate immunity to P. aeruginosa infection is multifactorial, with one key component being a specific ligand-receptor interaction between the protein and the microbe.     

Cystic fibrosis: a disease of vulnerability to airway surface dehydration

Cystic fibrosis (CF) lung disease involves chronic bacterial infection of retained airway secretions (mucus). Recent data suggest that CF lung disease pathogenesis reflects the vulnerability of airway surfaces to dehydration and collapse of mucus clearance. This predisposition is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in (i) the absence of CFTR-mediated Cl- secretion and regulation of epithelial Na+ channel (ENaC) function; and (ii) the sole dependence on extracellular ATP to rebalance these ion transport processes through P2 purinoceptor signaling. Recent clinical studies indicate that inhalation of hypertonic saline osmotically draws sufficient water onto CF airway surfaces to provide clinical benefit.     

Reevaluating gel-forming mucins' roles in cystic fibrosis lung disease

The existence of mucus plugs, containing mucins, bacteria, and neutrophils, blocking the lower airways in the lung of cystic fibrosis (CF) patients has raised the possibility that production of "abnormal" mucins is a critical characteristic of this disease. The molecular nature, if any, of this abnormality is unknown. Recent studies suggest that CF lung disease progression is characterized by an early phase in which airway surface liquid (ASL) increased dehydration is accompanied by altered pH and levels of reduced glutathione (GSH). In a later phase, bacterial infection and neutrophil invasion lead to increased ASL of concentrations myeloperoxidase and hypochlorous acid (HOCl). Independent studies indicate that gel-forming mucins, the key components of airway mucus, form disulfide-linked polymers through a pH-dependent, likely self-catalyzed mechanism. In this article, we present the hypothesis that increased mucus concentration (dehydration) and altered pH, and levels of GSH, myeloperoxidase, and/or HOCl result in the extracellular formation of additional interchain bonds among airway mucins. These novel interactions would create an atypical mucin network with abnormal viscoelastic and adhesive properties.     

Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes

The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properties, evaporative water loss, and the composition of secreted fluids. We developed a noninvasive approach to measure ASL osmolality using osmotically sensitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/polyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein was encapsulated in the liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a volume-insensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly (< 0.2 s) and stably to changes in solution osmolality. ASL osmolality was determined from calcein/sulforhodamine 101 fluorescence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 microm and osmolality was 325 +/- 12 mOsm. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 55 microm and osmolality was 330 +/- 36 mOsm. ASL osmolality was not affected by pharmacological inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to > 400 mOsm by exposure of the epithelium to dry air; the data were modeled mathematically using measured rates of osmosis and evaporative water loss. These results establish a ratio imaging method to map osmolality in biological compartments. ASL fluid is approximately isosmolar under normal physiological conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients.     

The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model

Asthma exacerbations can be triggered by viral infections or allergens. The Th2 cytokines IL-13 and IL-4 are produced during allergic responses and cause increases in airway epithelial cell mucus and electrolyte and water secretion into the airway surface liquid (ASL). Since ASL dehydration can cause airway inflammation and obstruction, ion transporters could play a role in pathogenesis of asthma exacerbations. We previously reported that expression of the epithelial cell anion transporter pendrin is markedly increased in response to IL-13. Herein we show that pendrin plays a role in allergic airway disease and in regulation of ASL thickness. Pendrin-deficient mice had less allergen-induced airway hyperreactivity and inflammation than did control mice, although other aspects of the Th2 response were preserved. In cultures of IL-13-stimulated mouse tracheal epithelial cells, pendrin deficiency caused an increase in ASL thickness, suggesting that reductions in allergen-induced hyperreactivity and inflammation in pendrin-deficient mice result from improved ASL hydration. To determine whether pendrin might also play a role in virus-induced exacerbations of asthma, we measured pendrin mRNA expression in human subjects with naturally occurring common colds caused by rhinovirus and found a 4.9-fold increase in mean expression during colds. Studies of cultured human bronchial epithelial cells indicated that this increase could be explained by the combined effects of rhinovirus and IFN-gamma, a Th1 cytokine induced during virus infection. We conclude that pendrin regulates ASL thickness and may be an important contributor to asthma exacerbations induced by viral infections or allergens.     

Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis

Efficient clearance of mucus and inhaled pathogens from the lung is dependent on an optimal airway surface liquid (ASL) volume, which is maintained by the regulated transport of sodium and chloride across the airway epithelium. Accumulating evidence suggests that impaired mucus clearance in cystic fibrosis (CF) airways is a result of ASL depletion caused by excessive Na(+) absorption through the epithelial sodium channel (ENaC). However, the cellular mechanisms that result in increased ENaC activity in CF airways are not completely understood. Recently, proteases were shown to modulate the activity of ENaC, but the relevance of this mechanism to the physiologic regulation of ASL volume is unknown. Using primary human airway epithelial cells, we demonstrate that: (i) protease inhibitors are present in the ASL and prevent the activation of near-silent ENaC, (ii) when the ASL volume is increased, endogenous protease inhibitors become diluted, allowing for proteolytic activation of near-silent channels, and (iii) in CF, the normally present near-silent pool of ENaC is constitutively active and the alpha subunit undergoes increased proteolytic processing. These findings indicate that the ASL volume modulates the activity of ENaC by modification of the serine protease-protease inhibitor balance and that alterations in this balance contribute to excessive Na(+) absorption in cystic fibrosis.     

Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl^- channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung’s innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca²⁺ in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca²⁺ largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca²⁺ elevation.     

Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl^- channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung’s innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca²⁺ in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca²⁺ largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca²⁺ elevation.     

Acinar origin of CFTR-dependent airway submucosal gland fluid secretion

Cystic fibrosis (CF) airway disease arises from defective innate defenses, especially defective mucus clearance of microorganisms. Airway submucosal glands secrete most airway mucus, and CF airway glands do not secrete in response to VIP or forskolin. CFTR, the protein that is defective in CF, is expressed in glands, but immunocytochemistry finds the highest expression of CFTR in either the ciliated ducts or in the acini, depending on the antibodies used. CFTR is absolutely required for forskolin-mediated gland secretion; we used this finding to localize the origin of forskolin-stimulated, CFTR-dependent gland fluid secretion. We tested the hypothesis that secretion to forskolin might originate from the gland duct rather than or in addition to the acini. We ligated gland ducts at various points, stimulated the glands with forskolin, and monitored the regions of the glands that swelled. The results supported an acinar rather than ductal origin of secretion. We tracked particles in the mucus using Nomarski time-lapse imaging; particles originated in the acini and traveled toward the duct orifice. Estimated bulk flow accelerated in the acini and mucus tubules, consistent with fluid secretion in those regions, but was constant in the unbranched duct, consistent with a lack of fluid secretion or absorption by the ductal epithelium. We conclude that CFTR-dependent gland fluid secretion originates in the serous acini. The failure to observe either secretion or absorption from the CFTR and epithelial Na(+) channel (ENaC)-rich ciliated ducts is unexplained, but may indicate that this epithelium alters the composition rather than the volume of gland mucus.     

Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells

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.     

The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition

Two hypotheses have been proposed recently that offer different views on the role of airway surface liquid (ASL) in lung defense. The "compositional" hypothesis predicts that ASL [NaCl] is kept low (<50 mM) by passive forces to permit antimicrobial factors to act as a chemical defense. The "volume" hypothesis predicts that ASL volume (height) is regulated isotonically by active ion transport to maintain efficient mechanical mucus clearance as the primary form of lung defense. To compare these hypotheses, we searched for roles for: (1) passive forces (surface tension, ciliary tip capillarity, Donnan, and nonionic osmolytes) in the regulation of ASL composition; and (2) active ion transport in ASL volume regulation. In primary human tracheobronchial cultures, we found no evidence that a low [NaCl] ASL could be produced by passive forces, or that nonionic osmolytes contributed substantially to ASL osmolality. Instead, we found that active ion transport regulated ASL volume (height), and that feedback existed between the ASL and airway epithelia to govern the rate of ion transport and volume absorption. The mucus layer acted as a "reservoir" to buffer periciliary liquid layer height (7 microm) at a level optimal for mucus transport by donating or accepting liquid to or from the periciliary liquid layer, respectively. These data favor the active ion transport/volume model hypothesis to describe ASL physiology.     

Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I^−/Cl⁻) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.