A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

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.

Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel.

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

Click-tambjamines as efficient and tunable bioactive anion transporters.

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design.

Small Molecule Anion Carriers Correct Abnormal Airway Surface Liquid Properties in Cystic Fibrosis Airway Epithelia.

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

KEY POINTS: Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. ABSTRACT: TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.

Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.

Small molecule-facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CF transmembrane conductance regulator (CFTR) protein, a cAMP-dependent anion channel involved in fluid transport across epithelium. Because small synthetic transmembrane anion transporters (anionophores) can replace the biological anion transport mechanisms, independent of genetic mutations in the CFTR, such anionophores are candidates as new potential treatments for CF. EXPERIMENTAL APPROACH: In order to assess their effects on cell physiology, we have analysed the transport properties of five anionophore compounds, three prodigiosines and two tambjamines. Chloride efflux was measured in large uni-lamellar vesicles and in HEK293 cells with chloride-sensitive electrodes. Iodide influx was evaluated in FRT cells transfected with iodide-sensitive YFP. Transport of bicarbonate was assessed by changes of pH after a NH4 + pre-pulse using the BCECF fluorescent probe. Assays were also carried out in FRT cells permanently transfected with wild type and mutant human CFTR. KEY RESULTS: All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores did not interfere with the activation of CFTR and did not modify the action of lumacaftor (a CFTR corrector) or ivacaftor (a CFTR potentiator). CONCLUSION AND IMPLICATIONS: These anionophores, at low concentrations, transported chloride and bicarbonate across cell membranes, without affecting CFTR function. They therefore provide promising starting points for the development of novel treatments for CF.

Peripheral localization of the epithelial sodium channel in the apical membrane of bronchial epithelial cells.

NEW FINDINGS: What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and ß ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+ /K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build-up in other regions of the cells.

SWATH label-free proteomics for cystic fibrosis research.

BACKGROUND: Label-free proteomics is a powerful tool for biological investigation. The SWATH protocol, relying on the Pan Human ion library, currently represents the state-of-the-art methodology for this kind of analysis. We recently discovered that this tool is not perfectly suitable for proteomics research in the CF field, as it lacks assays for several proteins crucial for the CF biology, including CFTR. METHODS: We extensively investigated the proteome of a very popular model for in vitro research on CF, CFBE41o-, and we used the corresponding data to improve the power of SWATH proteomics for CF investigation. We then used this improved tool to explore in depth the proteome of primary bronchial epithelial (BE) cells deriving from four CF individuals compared with that of four corresponding non-CF controls. By means of advanced bioinformatics tools, we outlined the presence of a number of protein networks being significantly altered by CF. RESULTS: Our analysis on patients' BE cells identified 154 proteins dysregulated by the CF pathology (94 upregulated and 60 downregulated). Some known CFTR interactors are present among them, but our analysis also revealed the alteration of other proteins not previously known to be related with CF. CONCLUSIONS: The present work outlines the power of SWATH label free proteomics applied to CF research.

In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia.

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.

Increased expression of ATP12A proton pump in cystic fibrosis airways.

Proton secretion mediated by ATP12A protein on the surface of the airway epithelium may contribute to cystic fibrosis (CF) lung disease by favoring bacterial infection and airway obstruction. We studied ATP12A in fresh bronchial samples and in cultured epithelial cells. In vivo, ATP12A expression was found almost exclusively at the apical side of nonciliated cells of airway epithelium and in submucosal glands, with much higher expression in CF samples. This could be due to bacterial infection and inflammation, since treating cultured cells with bacterial supernatants or with IL-4 (a cytokine that induces goblet cell hyperplasia) increased the expression of ATP12A in nonciliated cells. This observation was associated with upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A. ATP12A function was evaluated by measuring the pH of the apical fluid in cultured epithelia. Under resting conditions, CF epithelia showed more acidic values. This abnormality was minimized by inhibiting ATP12A with ouabain. Following treatment with IL-4, ATP12A function was markedly increased, as indicated by strong acidification occurring under bicarbonate-free conditions. Our study reveals potentially novel aspects of ATP12A and remarks its importance as a possible therapeutic target in CF and other respiratory diseases.

Anion-Transport Mechanism of a Triazole-Bearing Derivative of Prodigiosine: A Candidate for Cystic Fibrosis Therapy.

Cystic fibrosis (CF) is a genetic lethal disease, originated from the defective function of the CFTR protein, a chloride and bicarbonate permeable transmembrane channel. CF mutations affect CFTR protein through a variety of molecular mechanisms which result in different functional defects. Current therapeutic approaches are targeted to specific groups of patients that share a common functional defect. We seek to develop an innovative therapeutic approach for the treatment of CF using anionophores, small molecules that facilitate the transmembrane transport of anions. We have characterized the anion transport mechanism of a synthetic molecule based on the structure of prodigiosine, a red pigment produced by bacteria. Anionophore-driven chloride efflux from large unilamellar vesicles is consistent with activity of an uniporter carrier that facilitates the transport of anions through lipid membranes down the electrochemical gradient. There are no evidences of transport coupling with protons. The selectivity sequence of the prodigiosin inspired EH160 ionophore is formate > acetate > nitrate > chloride > bicarbonate. Sulfate, phosphate, aspartate, isothionate, and gluconate are not significantly transported by these anionophores. Protonation at acidic pH is important for the transport capacity of the anionophore. This prodigiosin derived ionophore induces anion transport in living cells. Its low toxicity and capacity to transport chloride and bicarbonate, when applied at low concentration, constitute a promising starting point for the development of drug candidates for CF therapy.

Pharmacological Inhibition of the Ubiquitin Ligase RNF5 Rescues F508del-CFTR in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability.

Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.

High-throughput screening identifies FAU protein as a regulator of mutant cystic fibrosis transmembrane conductance regulator channel.

In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism.

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR.

Murine Rankl-/- Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL-Expressing Lentiviral Vector.

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.

Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.