CFTR,Cell Junctions and the Cytoskeleton

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a cAMP regulated chloride (Cl⁻) and bicarbonate (HCO₃⁻) ion channel expressed at the apical plasma membrane (PM) of epithelial cells. Reduced CFTR protein results in decreased Cl⁻ secretion and excessive sodium reabsorption in epithelial cells, which consequently leads to epithelial dehydration and the accumulation of thick mucus within the affected organs, such as the lungs, pancreas, gastrointestinal (GI) tract, reproductive system and sweat glands. However, CFTR has been implicated in other functions besides transporting ions across epithelia. The rising number of references concerning its association to actin cytoskeleton organization, epithelial cell junctions and extracellular matrix (ECM) proteins suggests a role in the formation and maintenance of epithelial apical basolateral polarity. This review will focus on recent literature (the last 10 years) substantiating the role of CFTR in cell junction formation and actin cytoskeleton organization with its connection to the ECM.     

Pharmacological Effects of Panduratin A on Renal Cyst Development in In Vitro and In Vivo Models of Polycystic Kidney Disease

Renal cyst expansion in polycystic kidney disease (PKD) involves abnormalities in both cyst-lining-cell proliferation and fluid accumulation. Suppression of these processes may retard the progression of PKD. Evidence suggests that the activation of 5′ AMP-activated protein kinase (AMPK) inhibits cystic fibrosis transmembrane conductance regulator (CFTR)–mediated chloride secretion, leading to reduced progression of PKD. Here we investigated the pharmacological effects of panduratin A, a bioactive compound known as an AMPK activator, on CFTR-mediated chloride secretion and renal cyst development using in vitro and animal models of PKD. We demonstrated that AMPK was activated in immortalized normal renal cells and autosomal dominant polycystic kidney disease (ADPKD) cells following treatment with panduratin A. Treatment with panduratin A reduced the number of renal cyst colonies corresponding with a decrease in cell proliferation and phosphorylated p70/S6K, a downstream target of mTOR signaling. Additionally, panduratin A slowed cyst expansion via inhibition of the protein expression and transport function of CFTR. In heterozygous Han:Sprague–Dawley (Cy/+) rats, an animal model of PKD, intraperitoneal administration of panduratin A (25 mg/kg BW) for 5 weeks significantly decreased the kidney weight per body weight ratios and the cystic index. Panduratin A also reduced collagen deposition in renal tissue. Intraperitoneal administration of panduratin A caused abdominal bleeding and reduced body weight. However, 25 mg/kg BW of panduratin A via oral administration in the PCK rats, another non-orthologous PKD model, showed a significant decrease in the cystic index without severe adverse effects, indicating that the route of administration is critical in preventing adverse effects while still slowing disease progression. These findings reveal that panduratin A might hold therapeutic properties for the treatment of PKD.     

The Effect of CFTR Modulators on Airway Infection in Cystic Fibrosis

The advent of Cystic fibrosis transmembrane receptor (CFTR) modulators in 2012 was a critical event in the history of cystic fibrosis (CF) treatment. Unlike traditional therapies that target downstream effects of CFTR dysfunction, CFTR modulators aim to correct the underlying defect at the protein level. These genotype-specific therapies are now available for an increasing number of CF patients, transforming the way we view the condition from a life-limiting disease to one that can be effectively managed. Several studies have demonstrated the vast improvement CFTR modulators have on normalization of sweat chloride, CFTR function, clinical endpoints, and frequency of pulmonary exacerbation. However, their impact on other aspects of the disease, such as pathogenic burden and airway infection, remain under explored. Frequent airway infections as a result of increased susceptibility and impaired innate immune response are a serious problem within CF, often leading to accelerated decline in lung function and disease progression. Current evidence suggests that CFTR modulators are unable to eradicate pathogenic organisms in those with already established lung disease. However, this may not be the case for those with relatively low levels of disease progression and conserved microbial diversity, such as young patients. Furthermore, it remains unknown whether the restorative effects exerted by CFTR modulators extend to immune cells, such as phagocytes, which have the potential to modulate the response of people with CF (pwCF) to infection. Throughout this review, we look at the potential impact of CFTR modulators on airway infection in CF and their ability to shape impaired pulmonary defences to pathogens.     

A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator

The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.     

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.     

KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR

Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin–proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.     

Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX‐809 has been reported to facilitate the folding and trafficking of F508del‐CFTR and augment its channel function. The mechanism of action of VX‐809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX‐809: does it bind CFTR directly in order to exert its action? We synthesized two VX‐809 derivatives, ALK‐809 and SUL‐809, that possess an alkyne group and retain the rescue capacity of VX‐809. By using Cu^I‐catalyzed click chemistry, we provide evidence that the VX‐809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.     

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.     

Isorhamnetin Ameliorates Dry Eye Disease via CFTR Activation in Mice

Dry eye disease is one of the most common diseases, with increasing prevalence in many countries, but treatment options are limited. Cystic fibrosis transmembrane conductance regulator (CFTR) is a major ion channel that facilitates fluid secretion in ocular surface epithelium and is a potential target of therapeutic agent for the treatment of dry eye disease. In this study, we performed a cell-based, high-throughput screening for the identification of novel natural products that activate CFTR and restore the aqueous deficiency in dry eye. Screening of 1000 natural products revealed isorhamnetin, a flavonol aglycone, as a novel CFTR activator. Electrophysiological studies showed that isorhamnetin significantly increased CFTR chloride current, both wild type and ∆F508-CFTR. Isorhamnetin did not alter intracellular cAMP levels and the activity of other ion channels, including ANO1, ENaC, and hERG. Notably, application of isorhamnetin on mouse ocular surface induced CFTR activation and increased tear volume. In addition, isorhamnetin significantly reduced ocular surface damage and expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α in an experimental mouse model of dry eye. These data suggest that isorhamnetin may be used to treat dry eye disease.     

Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.     

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.     

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.     

Emerging Concepts in Defective Macrophage Phagocytosis in Cystic Fibrosis

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Chronic inflammation and decline in lung function are major reasons for morbidity in CF. Mutant CFTR expressed in phagocytic cells such as macrophages contributes to persistent infection, inflammation, and lung disease in CF. Macrophages play a central role in innate immunity by eliminating pathogenic microbes by a process called phagocytosis. Phagocytosis is required for tissue homeostasis, balancing inflammation, and crosstalk with the adaptive immune system for antigen presentation. This review focused on (1) current understandings of the signaling underlying phagocytic mechanisms; (2) existing evidence for phagocytic dysregulation in CF; and (3) the emerging role of CFTR modulators in influencing CF phagocytic function. Alterations in CF macrophages from receptor initiation to phagosome formation are linked to disease progression in CF. A deeper understanding of macrophages in the context of CFTR and phagocytosis proteins at each step of phagosome formation might contribute to the new therapeutic development of dysregulated innate immunity in CF. Therefore, the review also indicates future areas of research in the context of CFTR and macrophages.     

Increased Expression of Galectin-3 in Skin Fibrosis: Evidence from In Vitro and In Vivo Studies

Skin fibrosis is a hallmark of a wide array of dermatological diseases which can greatly impact the patients’ quality of life. Galectin-3 (GAL-3) has emerged as a central regulator of tissue fibrosis, playing an important pro-fibrotic role in numerous organs. Various studies are highlighting its importance as a skin fibrotic diseases biomarker; however, there is a need for further studies that clarify its role. This paper aims to ascertain whether the expression of GAL-3 is increased in relevant in vitro and in vivo models of skin fibrosis. We studied the role of GAL-3 in vitro using normal human dermal fibroblasts (NHDF) and fibrocytes. In addition, we used a skin fibrosis murine model (BALB/c mice) and human biopsies of healthy or keloid tissue. GAL-3 expression was analyzed using real time PCR, Western blot and immunostaining techniques. We report a significantly increased expression of GAL-3 in NHDF and fibrocytes cell cultures following stimulation with transforming growth factor β1 (TGFβ1). In vivo, GAL-3 expression was increased in a murine model of systemic sclerosis and in human keloid biopsies. In sum, this study underlines the involvement of GAL-3 in skin fibrosis using several models of the disease and highlights its role as a relevant target.     

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC–MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl⁻ transport in CF.     

Complementary Dual Approach for In Silico Target Identification of Potential Pharmaceutical Compounds in Cystic Fibrosis

Cystic fibrosis is a genetic disease caused by mutation of the CFTR gene, which encodes a chloride and bicarbonate transporter in epithelial cells. Due to the vast range of geno- and phenotypes, it is difficult to find causative treatments; however, small-molecule therapeutics have been clinically approved in the last decade. Still, the search for novel therapeutics is ongoing, and thousands of compounds are being tested in different assays, often leaving their mechanism of action unknown. Here, we bring together a CFTR-specific compound database (CandActCFTR) and systems biology model (CFTR Lifecycle Map) to identify the targets of the most promising compounds. We use a dual inverse screening approach, where we employ target- and ligand-based methods to suggest targets of 309 active compounds in the database amongst 90 protein targets from the systems biology model. Overall, we identified 1038 potential target–compound pairings and were able to suggest targets for all 309 active compounds in the database.     

Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer’s vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.     

In Vitro,Ex Vivo,and In Vivo Models for the Study of Pemphigus

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens.     

Oxidative Stress and Chemoradiation-Induced Oral Mucositis: A Scoping Review of In Vitro,In Vivo and Clinical Studies

Chemoradiation-induced mucositis is a debilitating condition of the gastrointestinal tract eventuating from antineoplastic treatment. It is believed to occur primarily due to oxidative stress mechanisms, which generate Reactive Oxygen Species (ROS). The aim of this scoping review was to assess the role of oxidative stress in the development of Oral Mucositis (OM). Studies from the literature, published in MEDLINE and SCOPUS, that evaluated the oxidative stress pathways or antioxidant interventions for OM, were retrieved to elucidate the current understanding of their relationship. Studies failing inclusion criteria were excluded, and those suitable underwent data extraction, using a predefined data extraction table. Eighty-nine articles fulfilled criteria, and these were sub-stratified into models of study (in vitro, in vivo, or clinical) for evaluation. Thirty-five clinical studies evaluated antioxidant interventions on OM’s severity, duration, and pain, amongst other attributes. A number of clinical studies sought to elucidate the protective or therapeutic effects of compounds that had been pre-determined to have antioxidant properties, without directly assessing oxidative stress parameters (these were deemed “indirect evidence”). Forty-seven in vivo studies assessed the capacity of various compounds to prevent OM. Findings were mostly consistent, reporting reduced OM severity associated with a reduction in ROS, malondialdehyde (MDA), myeloperoxidase (MPO), but higher glutathione (GSH) and superoxide dismutase (SOD) activity or expression. Twenty-one in vitro studies assessed potential OM therapeutic interventions. The majority demonstrated successful a reduction in ROS, and in select studies, secondary molecules were assessed to identify the mechanism. In summary, this review highlighted numerous oxidative stress pathways involved in OM pathogenesis, which may inform the development of novel therapeutic targets.