CFTR-dependent Chloride Secretion Research Articles

Additive Potentiation of R334W-CFTR Function by Novel Small Molecules.

The R334W (c.1000C>T, p.Arg334Trp) is a rare cystic fibrosis (CF)-causing mutation for which no causal therapy is currently approved. This mutation leads to a significant reduction of CF transmembrane conductance regulator (CFTR) channel conductance that still allows for residual function. Potentiators are small molecules that interact with CFTR protein at the plasma membrane to enhance CFTR-dependent chloride secretion, representing thus pharmacotherapies targeting the root cause of the disease. Here, we generated a new CF bronchial epithelial (CFBE) cell line to screen a collection of compounds and identify novel potentiators for R334W-CFTR. The active compounds were then validated by electrophysiological assays and their additive effects in combination with VX-770, genistein, or VX-445 were exploited in this cell line and further confirmed in intestinal organoids. Four compounds (LSO-24, LSO-25, LSO-38, and LSO-77) were active in the functional primary screen and their ability to enhance R334W-CFTR-dependent chloride secretion was confirmed using electrophysiological measurements. In silico ADME analyses demonstrated that these compounds follow Lipinski’s rule of five and are thus suggested to be orally bioavailable. Dose−response relationships revealed nevertheless suboptimal efficacy and weak potency exerted by these compounds. VX-770 and genistein also displayed a small potentiation of R334W-CFTR function, while VX-445 demonstrated no potentiator activity for this mutation. In the R334W-expressing cell line, CFTR function was further enhanced by the combination of LSO-24, LSO-25, LSO-38, or LSO-77 with VX-770, but not with genistein. The efficacy of potentiator VX-770 combined with active LSO compounds was further confirmed in intestinal organoids (R334W/R334W genotype). Taken together, these molecules were demonstrated to potentiate R334W-CFTR function by a different mechanism than that of VX-770. They may provide a feasible starting point for the design of analogs with improved CFTR-potentiator activity.

Phospholipase C controls chloride-dependent short-circuit current in human bronchial epithelial cells.

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCβ3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCβ3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCβ3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.

Ionocytes and CFTR Chloride Channel Expression in Normal and Cystic Fibrosis Nasal and Bronchial Epithelial Cells.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

Lipid Peroxidation Drives Renal Cyst Growth In Vitro through Activation of TMEM16A.

Transepithelial chloride- secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure. Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK in vitro cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts. Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts in vitro is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion in vitro. These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.

Roflumilast reverses CFTR-mediated ion transport dysfunction in cigarette smoke-exposed mice

BackgroundDysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction.MethodsA/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea.ResultsAcute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, −2.0 ± 0.4 mV, 131.3 ± 29.3 μA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 μA/cm2, P < 0.01 vs. vehicle −0.3 ± 0.7 mV, 10.4 ± 7.0 μA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, −15.22 ± 2.7 mV vs. air, −14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect.ConclusionsRoflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.

Characterization of mitochondrial function in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function

Evidence supporting the occurrence of oxidative stress in Cystic Fibrosis (CF) is well established and the literature suggests that oxidative stress is inseparably linked to mitochondrial dysfunction. Here, we have characterized mitochondrial function, in particular as it regards the steps of oxidative phosphorylation and ROS production, in airway cells either homozygous for the F508del-CFTR allele or stably expressing wt-CFTR. We find that oxygen consumption, ΔΨ generation, adenine nucleotide translocator-dependent ADP/ATP exchange and both mitochondrial Complex I and IV activities are impaired in CF cells, while both mitochondrial ROS production and membrane lipid peroxidation increase. Importantly, treatment of CF cells with the small molecules VX-809 and 4,6,4'-trimethylangelicin, which act as "correctors" for F508del CFTR by rescuing the F508del CFTR-dependent chloride secretion, while having no effect per sè on mitochondrial function in wt-CFTR cells, significantly improved all the above mitochondrial parameters towards values found in the airway cells expressing wt-CFTR. This novel study on mitochondrial bioenergetics provides a springboard for future research to further understand the molecular mechanisms responsible for the involvement of mitochondria in CF and identify the proteins primarily responsible for the F508del-CFTR-dependent mitochondrial impairment and thus reveal potential novel targets for CF therapy.

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization.

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion.

Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

Resveratrol Enhances Airway Surface Liquid Depth in Sinonasal Epithelium by Increasing Cystic Fibrosis Transmembrane Conductance Regulator Open Probability

BackgroundChronic rhinosinusitis engenders enormous morbidity in the general population, and is often refractory to medical intervention. Compounds that augment mucociliary clearance in airway epithelia represent a novel treatment strategy for diseases of mucus stasis. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed by the cystic fibrosis transmembrane conductance regulator (CFTR). The objectives of the present study were to test resveratrol, a strong potentiator of CFTR channel open probability, in preparation for a clinical trial of mucociliary activators in human sinus disease. MethodsPrimary sinonasal epithelial cells, immortalized bronchoepithelial cells (wild type and F508del CFTR), and HEK293 cells expressing exogenous human CFTR were investigated by Ussing chamber as well as patch clamp technique under non-phosphorylating conditions. Effects on airway surface liquid depth were measured using confocal laser scanning microscopy. Impact on CFTR gene expression was measured by quantitative reverse transcriptase polymerase chain reaction.ResultsResveratrol is a robust CFTR channel potentiator in numerous mammalian species. The compound also activated temperature corrected F508del CFTR and enhanced CFTR-dependent chloride secretion in human sinus epithelium ex vivo to an extent comparable to the recently approved CFTR potentiator, ivacaftor. Using inside out patches from apical membranes of murine cells, resveratrol stimulated an ~8 picosiemens chloride channel consistent with CFTR. This observation was confirmed in HEK293 cells expressing exogenous CFTR. Treatment of sinonasal epithelium resulted in a significant increase in airway surface liquid depth (in µm: 8.08+/-1.68 vs. 6.11+/-0.47,control,p<0.05). There was no increase CFTR mRNA. ConclusionResveratrol is a potent chloride secretagogue from the mucosal surface of sinonasal epithelium, and hydrates airway surface liquid by increasing CFTR channel open probability. The foundation for a clinical trial utilizing resveratrol as a therapeutic intervention to increase mucociliary transport and airway surface liquid hydration in sinus disease is strongly supported by these findings.

NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway

Tight junctions (TJs) restrict the transit of ions and molecules through the paracellular route and act as a barrier to regulate access of inflammatory cells into the airway lumen. The pathophysiology of cystic fibrosis (CF) lung disease is characterised by abnormal ion and fluid transport across the epithelium and polymorphonuclear (PMN) leukocyte-dominated inflammatory response. Na+/H+ exchanger regulatory factor 1 (NHERF1) is a protein involved in PKA-dependent activation of CFTR by interacting with CFTR via its PDZ domains and with ezrin via its C-terminal domain. We have previously found that the NHERF1-overexpression dependent rescue CFTR-dependent chloride secretion is due to the re-organisation of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1–RhoA–ezrin–actin. In this context, we here studied whether NHERF1 and CFTR are involved in the organisation and function of TJs. F508del CFBE41o− monolayers presented nuclear localisation of zonula occludens (ZO-1) and occludin as well as disorganisation of claudin 1 and junction-associated adhesion molecule 1 as compared with wild-type 16HBE14o− monolayers, paralleled by increased permeability to dextrans and PMN transmigration. Overexpression of either NHERF1 or CFTR in CFBE41o− cells rescued TJ proteins to their proper intercellular location and decreased permeability and PMN transmigration, while this effect was not achieved by overexpressing either NHERF1 deprived of ezrin-binding domain. Further, expression of a phospho-dead ezrin mutant, T567A, increased permeability in both 16HBE14o− cells and in a CFBE clone stably overexpressing NHERF1 (CFBE/sNHERF1), whereas a constitutively active form of ezrin, T567D, achieved the opposite effect in CFBE41o− cells. A dominant-negative form of RhoA (RhoA-N19) also disrupted ZO-1 localisation at the intercellular contacts dislodging it to the nucleus and increased permeability in CFBE/sNHERF1. The inhibitor Y27632 of Rho kinase (ROCK) increased permeability as well. Overall, these data suggest a significant role for the multiprotein complex CFTR–NHERF1–ezrin–actin in maintaining TJ organisation and barrier function, and suggest that the RhoA/ROCK pathway is involved.

Mo2042 Identification of a Novel CFTR Interactome in Contributing Diarrhea in Inflammatory Bowel Disease

Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay,macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.

Mo2043 Protein Kinase D1 (Pkd1) Activation is an Early Event in Wounded Monolayers of Intestinal Epithelial Cells

Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay,macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.

CFTR-Adenylyl Cyclase I Association Responsible for UTP Activation of CFTR in Well-Differentiated Primary Human Bronchial Cell Cultures

Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca(2+)-activated chloride channel (CaCC) is activated by Ca(2+) agonists like UTP. We found that most chloride current elicited by Ca(2+) agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca(2+) activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca(2+) agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca(2+) agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca(2+)/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1-CFTR association is responsible for Ca(2+)/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca(2+) agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.

Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis

BackgroundA challenging problem arising from cystic fibrosis (CF) newborn screening is the significant number of infants with hypertrypsinaemia (HIRT) with sweat chloride levels in the intermediate range and only one...

Na+/H+Exchanger Regulatory Factor 1 Overexpression-dependent Increase of Cytoskeleton Organization Is Fundamental in the Rescue of F508del Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway CFBE41o- Cells

We have demonstrated that Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Delta Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-DeltaERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Rescue of Functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by Vasoactive Intestinal Peptide in the Human Nasal Epithelial Cell Line JME/CF15

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

Chloride-Dependent Secretion of Alveolar Wall Liquid Determined by Optical-Sectioning Microscopy

The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr(-/-) mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr(-/-) mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease.

Assessment of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CFTR-null mice after bone marrow transplantation

Several studies have demonstrated that bone marrow (BM)-derived cells give rise to rare epithelial cells in the gastrointestinal (GI) and respiratory tracts after BM transplantation into myeloablated recipients. We investigate whether, after transplantation of cystic fibrosis transmembrane conductance regulator (CFTR)-positive BM-derived cells, BM-derived GI and airway epithelial cells can provide CFTR activity in the GI tract and nasal epithelium of recipient cystic fibrosis mice. CFTR-/- mice were transplanted with wild-type BM after receiving different doses of irradiation, and CFTR activity was assessed in vivo in individual mice over time by using rectal and nasal potential difference analyses and in vitro by Ussing chamber analysis. The data suggest that rare BM-derived epithelial cells in the GI and nasal epithelium detected in CFTR-/- transplanted mice provide a modest level of CFTR-dependent chloride secretion. Detection of CFTR mRNA and protein in tissues of transplanted CFTR-/- mice supports these data.

Na+/H+ Exchanger Regulatory Factor Isoform 1 Overexpression Modulates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Activity in Human Airway 16HBE14o- Cells and Rescues ΔF508 CFTR Functional Expression in Cystic Fibrosis Cells*

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTR-dependent chloride secretion.

1. In the human airway epithelium, VIP/PACAP receptors are distributed in nerve fibers and in epithelial cells but their role in transepithelial ion transport have not been reported. Here, we show that human bronchial epithelial Calu-3 cells expressed the VPAC(1) receptor subtype which shares similar high affinity for VIP and PACAP-27. 2. The stoichiometric binding parameters characterizing the (125)I-VIP and (125)I-PACAP-27 binding to these receptors were determined. 3. We found that VIP (EC(50) approximately 7.6 nM) and PACAP-27 (EC(50) approximately 10 nM) stimulated glibenclamide-sensitive and DIDS-insensitive iodide efflux in Calu-3 cells. 4. The protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, chelerythrine chloride prevented activation by both peptides demonstrating that PKA and PKC are part of the signaling pathway. This profile corresponds to the pharmacological signature of CFTR. 5. In the cystic fibrosis airway epithelial IB3-1 cell lacking functional CFTR but expressing VPAC(1) receptors, neither VIP, PACAP-27 nor forskolin stimulated chloride transport. 6. Ussing chamber experiments demonstrated stimulation of CFTR-dependent short-circuit currents by VIP or PACAP-27 applied to the basolateral but not to the apical side of Calu-3 cells monolayers. 7. This study shows the stimulation in human bronchial epithelial cells of CFTR-dependent chloride secretion following activation by VIP and PACAP-27 of basolateral VPAC(1) receptors.