Increasing Expression of the Normal Human CFTR cDNA in Cystic Fibrosis Epithelial Cells Results in a Progressive Increase in the Level of CFTR Protein Expression, but a Limit on the Level of cAMP-Stimulated Chloride Secretion

1994 ◽  
Vol 5 (9) ◽  
pp. 1121-1129 ◽  
Author(s):  
Melissa A. Rosenfeld ◽  
Stephen J. Rosenfeld ◽  
Claire Danel ◽  
Tyrone C. Banks ◽  
Ronald G. Crystal
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Chloride Secretion ◽  
Progressive Increase ◽  
Normal Human ◽  
Cftr Protein

CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells

1995 ◽  
Vol 269 (6) ◽  
pp. L855-L864 ◽  
Author(s):  
M. Mergey ◽  
M. Lemnaouar ◽  
D. Veissiere ◽  
M. Perricaudet ◽  
D. C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Gene Transfer ◽  
Epithelial Cells ◽  
De Novo ◽  
Tracheal Epithelial Cells ◽  
Normal Human ◽  
Cftr Protein ◽  
Tracheal Epithelial ◽  
And Control

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.

Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin

2001 ◽  
Vol 280 (5) ◽  
pp. C1031-C1037 ◽  
Author(s):  
Rangan Maitra ◽  
Collin M. Shaw ◽  
Bruce A. Stanton ◽  
Joshua W. Hamilton
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Chloride Secretion ◽  
Cell Surface Expression ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Δf508 Cftr ◽  
Cftr Protein

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

scholarly journals Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 861-870 ◽  
Author(s):  
A. Alev Gerçeker ◽  
Tanweer Zaidi ◽  
Peter Marks ◽  
David E. Golan ◽  
Gerald B. Pier
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bacterial Uptake ◽  
Cftr Protein ◽  
Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

Calcium-pump inhibitors induce functional surface expression of ΔF508-CFTR protein in cystic fibrosis epithelial cells

2002 ◽  
Vol 8 (5) ◽  
pp. 485-492 ◽  
Author(s):  
Marie E. Egan ◽  
Judith Glöckner-Pagel ◽  
Catherine A. Ambrose ◽  
Paula A. Cahill ◽  
Lamiko Pappoe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Surface Expression ◽  
Calcium Pump ◽  
Functional Surface ◽  
Δf508 Cftr ◽  
Cftr Protein

scholarly journals MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines

2011 ◽  
Vol 300 (1) ◽  
pp. L81-L87 ◽  
Author(s):  
Sharmistha Bhattacharyya ◽  
Usha Gutti ◽  
Jose Mercado ◽  
Chad Moore ◽  
Harvey B. Pollard ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Mrna Stability ◽  
Mapk Signaling ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
The Stability ◽  
Mapk Signaling Pathways

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3′-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

2005 ◽  
Vol 288 (5) ◽  
pp. L894-L902 ◽  
Author(s):  
James L. Kreindler ◽  
Alan D. Jackson ◽  
Philip A. Kemp ◽  
Robert J. Bridges ◽  
Henry Danahay
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Ion Transport ◽  
Cigarette Smoke ◽  
Apical Membrane ◽  
Chloride Secretion ◽  
Cigarette Smoke Extract ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.

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

2020 ◽  
Vol 21 (4) ◽  
pp. 1488 ◽  
Author(s):  
Ambra Gianotti ◽  
Valeria Capurro ◽  
Livia Delpiano ◽  
Marcin Mielczarek ◽  
María García-Valverde ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Fluid Composition ◽  
Airway Surface Liquid ◽  
Airway Epithelia ◽  
Drug Candidates ◽  
Cystic Fibrosis Airway ◽  
Functional Models ◽  
Cftr Protein ◽  
Surface Liquid

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.

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