Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting

Bruscia, Emanuela M.; Sangiuolo, Federica; Sinibaldi, P; Goncz, Kaarin K.; Novelli, Giuseppe; Gruenert, D. C.

doi:10.1038/sj.gt.3301741

Cited by 145 publications

(126 citation statements)

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“…Cell Lines and Cell Culture-Stable lentiviral-based transduction of the parental CFBE41oϪ cells (⌬F508/⌬F508), originally immortalized and characterized by Dr. D. Gruenert and co-workers (45,46) with either WT-CFTR or ⌬F508-CFTR, was performed by Tranzyme, Inc. (Birmingham, AL). The parental and stably transduced cells were generous gifts from Dr. J. P. Clancy (47).…”

Section: Methodsmentioning

confidence: 99%

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

Swiatecka‐Urban

Brown

Moreau‐Marquis

et al. 2005

Journal of Biological Chemistry

170

192

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The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl ؊ channels in the apical plasma membrane. Rescue of ⌬F508-CFTR by reduced temperature or chemical means reveals that the ⌬F508 mutation reduces the half-life of ⌬F508-CFTR in the apical plasma membrane. Because ⌬F508-CFTR retains some Cl ؊ channel activity, increased expression of ⌬F508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ⌬F508-CFTR that lead to the decreased apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o؊) the ⌬F508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is an ATP binding cassette (ABC) transporter and a cAMP-regulated Cl Ϫ channel that mediates transepithelial Cl Ϫ transport in the airways, intestine, pancreas, testis, and other tissues (1-3). Cystic fibrosis (CF), a lethal genetic disease, is caused by mutations in the CFTR gene (1, 2). The most common mutation in CFTR is ⌬F508 (4, 5). ⌬F508-CFTR does not fold properly, and most of the protein is retained within the endoplasmic reticulum (ER) where it is subsequently degraded (5, 6). Several studies suggest that the ER retention of ⌬F508-CFTR is not complete, and some ⌬F508-CFTR is constitutively expressed in the plasma membrane of primary epithelial cells from individuals homozygous for the ⌬F508 mutation (7-10). Because ⌬F508-CFTR retains some Cl Ϫ channel activity when expressed in the plasma membrane (5,6,(11)(12)(13)(14), it would be desirable to increase the expression of ⌬F508-CFTR in the plasma membrane to alleviate the symptoms in CF patients. The trafficking of ⌬F508-CFTR to the plasma membrane can be increased by chemical means or reduced temperature (15-21). Yet, functional and biochemical studies in heterologous cell lines demonstrate that rescued ⌬F508-CFTR has a greatly reduced stability or halflife in the post-ER compartments, including the plasma membrane (13,(22)(23)(24). Very little is known about the apical membrane half-life of rescued ⌬F508-CFTR in polarized human airway epithelial cells. A recent study demonstrates that the functional stability of ⌬F508-CFTR in the apical membrane of differentiated respiratory epithelial cells derived from nasal polyps from individuals homozygous for the ⌬F508 mutation is decreased compared with WT-CFTR (25). Furthermore, the bioc...

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Section: Methodsmentioning

confidence: 99%

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

Swiatecka‐Urban

Brown

Moreau‐Marquis

et al. 2005

Journal of Biological Chemistry

170

192

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“…− , 9HTEo-/CFTE29o − and S9/IB3 epithelial cell studies The human non-CF 16HBE14o − and 9HTEo − and F508del homozygous CFBE41o − and CFTE29o − bronchial and tracheal epithelial cell lines were supplied as a gift from D. Gruenert (California Pacific Medical Centre Research Institute, San Francisco, CA, USA) [28,29]. IB3 cells are a CF bronchial epithelial cell line (F508del/W1282X), S9s are their isogenic non-CF counterpart and both were obtained from Pamela Zeitlin ( Johns Hopkins Children's Centre, Baltimore, MD, USA) [30].…”

Section: Hbe14o-/cfbe41omentioning

confidence: 99%

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

et al. 2015

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Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis.IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured.miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o− tracheal, CFBE41o− and/or IB3 bronchial epithelial cells.These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

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“…Immortalized human bronchial epithelial cells 16HBE14o(HBE) (36) and immortalized bronchial epithelial cells from a (DF508/DF508) CF patient CFBE41o (CFBE) (37) were kindly donated by Dr. D.C. Gruenert (University of California, San Francisco, San Francisco, CA). Cells were grown in flasks coated with fibronectin (1 mg/ml), collagen (1 mg/ml; BD Biosciences, Bedford, MA), and BSA (1 mg/ml) in complete media (MEM containing 10% [v/v] heat-inactivated FCS and 1% [v/v] penicillin/ streptomycin) at 37˚C in a humidified incubator with 5% CO 2 .…”

Section: Cell Culturementioning

confidence: 99%

Dysregulation of TIM-3–Galectin-9 Pathway in the Cystic Fibrosis Airways

Vega-Carrascal

Reeves

Niki

et al. 2011

The Journal of Immunology

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The T-cell Ig and mucin domain-containing molecules (TIMs) have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. It has been reported that proinflammatory cytokine dysregulation and neutrophil-dominated inflammation are the main causes of morbidity in cystic fibrosis (CF). However, the role of TIM receptors in CF has not been investigated. In this study, we demonstrated that TIM-3 is constitutively overexpressed in the human CF airway, suggesting a link between CF transmembrane conductance regulator (CFTR) function and TIM-3 expression. Blockade of CFTR function with the CFTR inhibitor-172 induced an upregulation of TIM-3 and its ligand galectin-9 in normal bronchial epithelial cells. We also established that TIM-3 serves as a functional receptor in bronchial epithelial cells, and physiologically relevant concentrations of galectin-9 induced TIM-3 phosphorylation, resulting in increased IL-8 production. In addition, we have demonstrated that both TIM-3 and galectin-9 undergo rapid proteolytic degradation in the CF lung, primarily because of neutrophil elastase and proteinase-3 activity. Our results suggest a novel intrinsic defect that may contribute to the neutrophil-dominated immune response in the CF airways.

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Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting

Abstract: Cystic fibrosis is the most common inherited disease in the

Cited by 145 publications

References 4 publications

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

Dysregulation of TIM-3–Galectin-9 Pathway in the Cystic Fibrosis Airways

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