Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTR

Matos, Ana I.; Gomes-Duarte, Andreia; Faria, Márcia; Barros, Patrícia; Jordan, Peter; Amaral, Margarida D.; Matos, Paulo

doi:10.1038/s41598-018-31514-2

Cited by 22 publications

(36 citation statements)

References 64 publications

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“…Moreover, whereas the authors did not fully elucidate the mechanism of PM CFTR destabilization in PBMCs (44), here we show that knockdown of CAPN1 stabilizes Phe508del-CFTR by promoting the association of endogenous EZR with the CFTR-NHERF1 complex at the PM. This result is consistent with our previous findings showing that it is possible to coax the anchoring and PM retention of chemically rescued Phe508del-CFTR by promoting the activation and association of EZR to CFTR-NHERF1 complexes at the PM, by increasing endogenous RAC1 signaling through the stimulation of airway epithelial cells with HGF (28,42). The binding of activated EZR induces a conformational change in NHERF1 exposing a second PDZ domain for CFTR binding, which stabilizes the rescued channel at the cell surface, leading to a 3-fold increase in the functional restoration achieved by corrector drugs, such as VX-809 (28,29,42).…”

Section: Capn1 Binding Reduces Rphe508del-cftr Pm Stabilitysupporting

confidence: 93%

“…Having previously shown that rPhe508del-CFTR destabilization at the cell surface results from an interference with the formation of the CFTR-NHERF1-EZR retention complex at the PM (28,29,42), we further restricted this network to proteins annotated as direct interactors of either EZR, NHERF1, or CFTR. This approach generated a subnetwork of 22 proteins ( Fig.…”

Section: Capn1 Binding Reduces Rphe508del-cftr Pm Stabilitymentioning

confidence: 99%

“…After background subtraction, HS-YFP fluorescence recordings (F) were normalized to the initial average value measured before addition of I Ϫ (F 0 ). Quantification of fluorescence decay was performed on at least 24 individual cells/well, using ImageJ (National Institutes of Health) as previously described (42). The average fluorescence decay was fitted to an exponential decay function to derive the maximal slope that corresponds to initial influx of I Ϫ into the cells.…”

Section: Cftr Functional Assay By Hs-yfpmentioning

confidence: 99%

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Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant

Matos

Pinto

Barros

et al. 2019

Journal of Biological Chemistry

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Edited by Roger J. Colbran Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na ؉ /H ؉-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR-or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function. The cystic fibrosis (CF) 2 transmembrane conductance regulator (CFTR) is a multispanning cAMP-regulated chloride channel primarily localized to the apical membrane of polarized epithelial cells (1). Mutations in the CFTR gene cause the complex inherited disorder CF (1, 2). The most common CFTR mutation is the deletion of phenylalanine 508 (Phe508del), with ϳ85% of all CF patients having at least one copy of the Phe508del mutant (Welcome to CFTR2 website, https://www.cftr2.org/) 3 (4). This mutation is mainly characterized by protein misfolding and premature degradation by the endoplasmic reticulum (ER) quality control, preventing the mutant protein from trafficking to the cell surface (5-7). Although Phe508del ER retention is not complete, the very small fraction of channels that reach the cell surface possesses only partial activity because of a gating defect (5, 8, 9) and show a severely decreased plasma membrane (PM) halflife, because of accelerated endocytosis and lysosomal degradation (10, 11). At present, there are two approved corrector drugs that target the molecular defects in Phe508del (12-14). The first to be used in the clinic is the drug Orkambi, which combines "corrector" VX-809 (lumacaftor) and "potentiato...

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Section: Capn1 Binding Reduces Rphe508del-cftr Pm Stabilitysupporting

confidence: 93%

Section: Capn1 Binding Reduces Rphe508del-cftr Pm Stabilitymentioning

confidence: 99%

Section: Cftr Functional Assay By Hs-yfpmentioning

confidence: 99%

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Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant

Matos

Pinto

Barros

et al. 2019

Journal of Biological Chemistry

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“…Coadministration of HGF and lumacaftor has further enhanced CFTR maturation and anchoring at the PM (Loureiro et al, 2015). Prolonged HGF treatment also prevented ivacaftormediated destabilization of lumacaftor-rescued CFTR in F508del-expressing cells (Matos et al, 2018). Other strategies have demonstrated to enhance the PM stabilization of mutant CFTR protein, including administration of vasoactive intestine peptide (VIP) (Alshafie et al, 2014), activation of exchange factor directly activated by cAMP 1 (EPAC1) (Lobo et al, 2016), and inhibition of S-nitrosoglutathione reductase (Zaman et al, 2016) or CFTR-associated ligand (CAL) (Cusing et al, 2010;Bergbower et al, 2018).…”

Section: Stabilizers: Rescuing the Protein Stability At The Plasma Mementioning

confidence: 89%

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

2020

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Cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which result in impairment of CFTR mRNA and protein expression, function, stability or a combination of these. Although CF leads to multifaceted clinical manifestations, the respiratory disorder represents the major cause of morbidity and mortality of these patients. The life expectancy of CF patients has substantially lengthened due to early diagnosis and improvements in symptomatic therapeutic regimens. Quality of life remains nevertheless limited, as these individuals are subjected to considerable clinical, psychosocial and economic burdens. Since the discovery of the CFTR gene in 1989, tremendous efforts have been made to develop therapies acting more upstream on the pathogenesis cascade, thereby overcoming the underlying dysfunctions caused by CFTR mutations. In this line, the advances in cell-based high-throughput screenings have been facilitating the fast-tracking of CFTR modulators. These modulator drugs have the ability to enhance or even restore the functional expression of specific CF-causing mutations, and they have been classified into five main groups depending on their effects on CFTR mutations: potentiators, correctors, stabilizers, read-through agents, and amplifiers. To date, four CFTR modulators have reached the market, and these pharmaceutical therapies are transforming patients' lives with short-and long-term improvements in clinical outcomes. Such breakthroughs have paved the way for the development of novel CFTR modulators, which are currently under experimental and clinical investigations. Furthermore, recent insights into the CFTR structure will be useful for the rational design of next-generation modulator drugs. This review aims to provide a summary of recent developments in CFTR-directed therapeutics. Barriers and future directions are also discussed in order to optimize treatment adherence, identify feasible and sustainable solutions for equitable access to these therapies, and continue to expand the pipeline of novel modulators that may result in effective precision medicine for all individuals with CF.

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“…Thus, acute co-treatment with HGF can significantly enhance the chemical correction of Phe508del-CFTR via induction of RAC1. Interestingly, prolonged, 15-day HGF treatment also significantly improves the functional rescue of Phe508del-CFTR by Ivacaftor/Lumacaftor in polarized bronchial epithelial monolayers 33 .…”

Section: Discussionmentioning

confidence: 92%

Plasma microRNA levels in male and female children with cystic fibrosis

Mooney

McKiernan

Raoof

et al. 2020

Sci Rep

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A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1-6; Median Age: 3; 9 p.Phe508del homo-or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p < 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plasma of females versus males with CF under six years of age. Cystic fibrosis (CF) is an autosomal recessive genetic disorder arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene 1. CFTR is a crucial chloride ion channel expressed by epithelial cells in the lung and other organs. The consequences of defective CFTR are most severely manifested in the lung, where the imbalanced ion homeostasis results in chronic airway inflammation, which leads to a progressive and ultimately fatal decline in lung function. Despite improved therapies and increased life expectancy for both males and females with CF in recent years, a gender dichotomy persists whereby females are at a clinical disadvantage e.g. poorer lung function, earlier colonisation by chronic mucoid Pseudomonas aeruginosa, greater frequency of exacerbations, and lower median survival age 2-7. Whether sex differences exist in children with CF remains controversial 8,9. The ability to measure and monitor lung disease in children via a non-invasive method would be highly beneficial for CF clinical care. Molecular biomarkers that are prognostic for lung disease progression could serve, in support of clinical examination, to allow earlier and hence more effective intervention strategies. Ideally these would be molecules that are stable in body fluids (e.g. plasma), able to be quantified with reliance and accuracy, and mechanistically linked to pulmonary infection and/or inflammation. MicroRNAs are small non-coding RNAs that bind specific target mRNA sequences to inhibit their expression. As well as being expressed within cells/tissues, miRNAs are also present extracellularly, and are readily detectable in body fluids such as serum and plasma. In contrast to mRNA, miRNAs circulating in plasma are surprisingly resistant to RNase degradation due to the fact they are found either packaged inside microvesicles e.g. exosomes, or associated with protein/lipid complexes e.g. argonaute 2 or high density lipoprotein 10. Although the role of circulating miRNAs is not...

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Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTR

Cited by 22 publications

References 64 publications

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

Plasma microRNA levels in male and female children with cystic fibrosis

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