mRNA Delivery: Challenges and Advances through Polymeric Soft Nanoparticles.

Single-stranded messenger ribonucleic acid (mRNA) plays a pivotal role in transferring genetic information, and tremendous effort has been devoted over the years to utilize its transcription efficacy in therapeutic interventions for a variety of diseases with high morbidity and mortality. Lipid nanocarriers have been extensively investigated for mRNA delivery and enabled the rapid and successful development of mRNA vaccines against SARS-CoV-2. Some constraints of lipid nanocarriers have encouraged the development of alternative delivery systems, such as polymer-based soft nanoparticles, which offer a modular gene delivery platform. Such macromolecule-based nanocarriers can be synthetically articulated for tailored parameters including mRNA protection, loading efficacy, and targeted release. In this review, we highlight recent advances in the development of polymeric architectures for mRNA delivery, their limitations, and the challenges that still exist, with the aim of expediting further research and the clinical translation of such formulations.

CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

Ionocyte-Specific Regulation of Cystic Fibrosis Transmembrane Conductance Regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

Chronic obstructive pulmonary disease and the modulation of CFTR by acute exposure to cigarette smoke.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death and cigarette smoke is the main risk factor. Detecting its earliest stages and preventing a decline in lung function are key goals. The pathogenesis of COPD is complex but has some similarities to cystic fibrosis (CF), a disease caused by mutations in the cftr gene. CF leads to chronic inflammation, abnormal mucus, and cycles of infection. Cigarette smoke exposure also causes CFTR dysfunction, and it is probably not a coincidence that inflammation, mucus obstruction, and infections are also characteristics of COPD, although the exacerbations can be quite different. We review here the acute effects of cigarette smoke on CFTR function and its potential role in COPD. Understanding CFTR regulation by cigarette smoke may identify novel drug targets and facilitate the development of therapeutics that reduce the progression and severity of COPD.

Treatment With LAU-7b Complements CFTR Modulator Therapy by Improving Lung Physiology and Normalizing Lipid Imbalance Associated With CF Lung Disease.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasians, affecting more than 100,000 individuals worldwide. It is caused by pathogenic variants in the gene encoding CFTR, an anion channel at the plasma membrane of epithelial and other cells. Many CF pathogenic variants disrupt the biosynthesis and trafficking of CFTR or reduce its ion channel function. The most frequent mutation, loss of a phenylalanine at position 508 (F508del), leads to misfolding, retention in the endoplasmic reticulum, and premature degradation of the protein. The therapeutics available for treating CF lung disease include antibiotics, mucolytics, bronchodilators, physiotherapy, and most recently CFTR modulators. To date, no cure for this life shortening disease has been found. Treatment with the Triple combination drug therapy, TRIKAFTA®, is composed of three drugs: Elexacaftor (VX-445), Tezacaftor (VX-661) and Ivacaftor (VX-770). This therapy, benefits persons with CF, improving their weight, lung function, energy levels (as defined by reduced fatigue), and overall quality of life. We examined the effect of combining LAU-7b oral treatment and Triple therapy combination on lung function in a F508deltm1EUR mouse model that displays lung abnormalities relevant to human CF. We assessed lung function, lung histopathology, protein oxidation, lipid oxidation, and fatty acid and lipid profiles in F508deltm1EUR mice.

Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR.

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809) and the triple combination Trikafta®. This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in cystic fibrosis therapeutics.

The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway.

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane. This article has an associated First Person interview with the first author of the paper.

Rescue of Mutant CFTR Trafficking Defect by the Investigational Compound MCG1516A.

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.

Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells.

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. METHODS: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. RESULTS: PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. CONCLUSION: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium.

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

Cyclic nucleotide phosphodiesterase inhibitors as therapeutic interventions for cystic fibrosis.

Cystic Fibrosis (CF) lung disease results from mutations in the CFTR anion channel that reduce anion and fluid secretion by airway epithelia. Impaired secretion compromises airway innate defence mechanisms and leads to bacterial colonization, excessive inflammation and tissue damage; thus, restoration of CFTR function is the goal of many CF therapies. CFTR channels are activated by cyclic nucleotide-dependent protein kinases. The second messengers 3'5'-cAMP and 3'5'-cGMP are hydrolysed by a large family of cyclic nucleotide phosphodiesterases that provide subcellular spatial and temporal control of cyclic nucleotide-dependent signalling. Selective inhibition of these enzymes elevates cyclic nucleotide levels, leading to activation of CFTR and other downstream effectors. Here we examine members of the PDE family that are likely to regulate CFTR-dependent ion and fluid secretion in the airways and discuss other actions of PDE inhibitors that can influence cyclic nucleotide-regulated mucociliary transport, inflammation and bronchodilation. Finally, we review PDE inhibitors and the potential benefits they could provide as CF therapeutics.

Oxygenation as a driving factor in epithelial differentiation at the air-liquid interface.

Culture at the air-liquid interface is broadly accepted as necessary for differentiation of cultured epithelial cells towards an in vivo-like phenotype. However, air-liquid interface cultures are expensive, laborious and challenging to scale for increased throughput applications. Deconstructing the microenvironmental parameters that drive these differentiation processes could circumvent these limitations, and here we hypothesize that reduced oxygenation due to diffusion limitations in liquid media limits differentiation in submerged cultures; and that this phenotype can be rescued by recreating normoxic conditions at the epithelial monolayer, even under submerged conditions. Guided by computational models, hyperoxygenation of atmospheric conditions was applied to manipulate oxygenation at the monolayer surface. The impact of this rescue condition was confirmed by assessing protein expression of hypoxia-sensitive markers. Differentiation of primary human bronchial epithelial cells isolated from healthy patients was then assessed in air-liquid interface, submerged and hyperoxygenated submerged culture conditions. Markers of differentiation, including epithelial layer thickness, tight junction formation, ciliated surface area and functional capacity for mucociliary clearance, were assessed and found to improve significantly in hyperoxygenated submerged cultures, beyond standard air-liquid interface or submerged culture conditions. These results demonstrate that an air-liquid interface is not necessary to produce highly differentiated epithelial structures, and that increased availability of oxygen and nutrient media can be leveraged as important strategies to improve epithelial differentiation for applications in respiratory toxicology and therapeutic development.

Large pH oscillations promote host defense against human airways infection.

The airway mucosal microenvironment is crucial for host defense against inhaled pathogens but remains poorly understood. We report here that the airway surface normally undergoes surprisingly large excursions in pH during breathing that can reach pH 9.0 during inhalation, making it the most alkaline fluid in the body. Transient alkalinization requires luminal bicarbonate and membrane-bound carbonic anhydrase 12 (CA12) and is antimicrobial. Luminal bicarbonate concentration and CA12 expression are both reduced in cystic fibrosis (CF), and mucus accumulation both buffers the pH and obstructs airflow, further suppressing the oscillations and bacterial-killing efficacy. Defective pH oscillations may compromise airway host defense in other respiratory diseases and explain CF-like airway infections in people with CA12 mutations.

CFTR Correctors and Antioxidants Partially Normalize Lipid Imbalance but not Abnormal Basal Inflammatory Cytokine Profile in CF Bronchial Epithelial Cells.

A deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) function in CF leads to chronic lung disease. CF is associated with abnormalities in fatty acids, ceramides, and cholesterol, their relationship with CF lung pathology is not completely understood. Therefore, we examined the impact of CFTR deficiency on lipid metabolism and pro-inflammatory signaling in airway epithelium using mass spectrometric, protein array. We observed a striking imbalance in fatty acid and ceramide metabolism, associated with chronic oxidative stress under basal conditions in CF mouse lung and well-differentiated bronchial epithelial cell cultures of CFTR knock out pig and CF patients. Cell-autonomous features of all three CF models included high ratios of ω-6- to ω-3-polyunsaturated fatty acids and of long- to very long-chain ceramide species (LCC/VLCC), reduced levels of total ceramides and ceramide precursors. In addition to the retinoic acid analog fenretinide, the anti-oxidants glutathione (GSH) and deferoxamine partially corrected the lipid profile indicating that oxidative stress may promote the lipid abnormalities. CFTR-targeted modulators reduced the lipid imbalance and oxidative stress, confirming the CFTR dependence of lipid ratios. However, despite functional correction of CF cells up to 60% of non-CF in Ussing chamber experiments, a 72-h triple compound treatment (elexacaftor/tezacaftor/ivacaftor surrogate) did not completely normalize lipid imbalance or oxidative stress. Protein array analysis revealed differential expression and shedding of cytokines and growth factors from CF epithelial cells compared to non-CF cells, consistent with sterile inflammation and tissue remodeling under basal conditions, including enhanced secretion of the neutrophil activator CXCL5, and the T-cell activator CCL17. However, treatment with antioxidants or CFTR modulators that mimic the approved combination therapies, ivacaftor/lumacaftor and ivacaftor/tezacaftor/elexacaftor, did not effectively suppress the inflammatory phenotype. We propose that CFTR deficiency causes oxidative stress in CF airway epithelium, affecting multiple bioactive lipid metabolic pathways, which likely play a role in CF lung disease progression. A combination of anti-oxidant, anti-inflammatory and CFTR targeted therapeutics may be required for full correction of the CF phenotype.

The Phosphodiesterase Inhibitor Ensifentrine Reduces Production of Proinflammatory Mediators in Well Differentiated Bronchial Epithelial Cells by Inhibiting PDE4.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of proinflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function; thus, there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects; therefore, we examined whether ensifentrine alters the production of proinflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 and granulocyte monocyte colony-stimulating factor (GM-CSF) during challenge with interleukin-1ß Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors, respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the ß 2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate that ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with ß 2-adrenergic agonists or corticosteroids. SIGNIFICANCE STATEMENT: Airway inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the cystic fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of proinflammatory factors in well differentiated CF bronchial epithelial cells that is further enhanced when combined with ß2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with ß2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.

Characterization of the mechanism of action of RDR01752, a novel corrector of F508del-CFTR.

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

The dual phosphodiesterase 3/4 inhibitor RPL554 stimulates rare class III and IV CFTR mutants.

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

Changes in the R-region interactions depend on phosphorylation and contribute to PKA and PKC regulation of the cystic fibrosis transmembrane conductance regulator chloride channel.

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced by PKC phosphorylation, our data support a model in which the regulation of CFTR activation results from increased interactions of the R-region with the N-term-TMD1-NBD1. Also, serine S686 was found to be critical for the PKC enhancing effect which requires a permissive BH-R interaction at basal level and increased FH-R interaction after PKC + PKA phosphorylation.

Fenretinide favorably affects mucins (MUC5AC/MUC5B) and fatty acid imbalance in a manner mimicking CFTR-induced correction.

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome. Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.