scholarly journals Vardenafil increases intracellular accumulation of the most prevalent mutant cystic fibrosis transmembrane conductance regulator (CTFR) in human bronchial epithelial cells

Biology Open ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. bio053116
Author(s):  
Barbara Dhooghe ◽  
Caroline Bouzin ◽  
Angélique Mottais ◽  
Emmanuel Hermans ◽  
Martial Delion ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Lines ◽  
Chloride Transport ◽  
Pde5 Inhibitor ◽  
Inhibitory Effect ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Transient Rise ◽  
Phosphodiesterase Type ◽  
Cf Transmembrane Conductance Regulator

ABSTRACTCystic fibrosis (CF) is a genetic disease characterized by progressive lung and chronic digestive manifestations. We have shown that therapeutic doses of vardenafil, a phosphodiesterase type 5 (PDE5) inhibitor, corrects CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport in respiratory and intestinal tissues of F508del homozygous mice. Here, we studied the effect of vardenafil on CFTR in 16HBE14o– and CFBE41o− cell lines. First, the expression levels of PDE5 mRNA in these cell lines were monitored. The two cell lines were exposed to different drugs (dimethyl sulfoxide, 8-Br-cGMP, forskolin or vardenafil). The cAMP and cGMP intracellular concentrations were measured. Finally, we localised the CFTR by immunolabelling. PDE5 was similarly expressed in both wild-type and in CF cells. A fast and transient rise in cGMP intracellular contents followed treatment with vardenafil, confirming its PDE5 inhibitory effect. We showed that vardenafil promoted both the early steps of the cellular processing and the trafficking of F508del without fully addressing the protein to the plasma membrane. The effect was not reproduced by the brominated cGMP analogue and it was not prevented by the combination of a protein kinase G (PKG) inhibitor and vardenafil. These findings support the view that vardenafil partially rescues F508del through cGMP/PKG-independent mechanisms.

scholarly journals VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

2013 ◽  
Vol 24 (19) ◽  
pp. 3016-3024 ◽  
Author(s):  
Hong Yu Ren ◽  
Diane E. Grove ◽  
Oxana De La Rosa ◽  
Scott A. Houck ◽  
Pattarawut Sopha ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Genetic Disorder ◽  
Missense Mutations ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Regulator Protein ◽  
Folding Defect ◽  
Membrane Spanning ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

scholarly journals microRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene

2011 ◽  
Vol 438 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Austin E. Gillen ◽  
Nehal Gosalia ◽  
Shih-Hsing Leir ◽  
Ann Harris
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Chloride Transport ◽  
Intestinal Cell ◽  
Transmembrane Conductance Regulator ◽  
Regulator Gene ◽  
Transmembrane Conductance ◽  
General Role ◽  
Cystic Fibrosis Transmembrane

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3′ UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3′ UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na+–K+–Cl− co-transporter SLC12A2 [solute carrier family 12 (Na+–K+–Cl− transporters), member 2] 3′ UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

1997 ◽  
Vol 273 (5) ◽  
pp. L1065-L1072 ◽  
Author(s):  
Thomas J. Kelley ◽  
Calvin U. Cotton ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Nasal Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cyclic Monophosphate ◽  
Δf508 Cftr ◽  
Membrane Bound

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl− transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl− secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.

Examining basal chloride transport using the nasal potential difference response in a murine model

2001 ◽  
Vol 281 (5) ◽  
pp. L1173-L1179 ◽  
Author(s):  
Kristine G. Brady ◽  
Thomas J. Kelley ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Inbred Mice ◽  
Inbred Strains ◽  
Potential Difference ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Inbred Strains Of Mice ◽  
Cystic Fibrosis Transmembrane

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate ( Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.

scholarly journals Influence of Cystic Fibrosis Transmembrane Conductance Regulator on Gene Expression in Response to Pseudomonas aeruginosa Infection of Human Bronchial Epithelial Cells

2005 ◽  
Vol 73 (10) ◽  
pp. 6822-6830 ◽  
Author(s):  
Nina Reiniger ◽  
Jeffrey K. Ichikawa ◽  
Gerald B. Pier
Keyword(s):  
Cystic Fibrosis ◽  
Cell Line ◽  
Cell Lines ◽  
The Other ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Bronchial Epithelial ◽  
Cystic Fibrosis Transmembrane ◽  
In Cells

ABSTRACT Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

scholarly journals Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

1991 ◽  
Vol 11 (8) ◽  
pp. 3886-3893 ◽  
Author(s):  
R J Gregory ◽  
D P Rich ◽  
S H Cheng ◽  
D W Souza ◽  
S Paul ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Amino Acid ◽  
Functional Activity ◽  
Nucleotide Binding ◽  
Missense Mutations ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Fluorescence Measurements ◽  
Cf Transmembrane Conductance Regulator ◽  
Altered Proteins

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

scholarly journals Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences

2009 ◽  
Vol 297 (4) ◽  
pp. L677-L686 ◽  
Author(s):  
R. William Vandivier ◽  
Tiffany R. Richens ◽  
Sarah A. Horstmann ◽  
Aimee M. deCathelineau ◽  
Moumita Ghosh ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rho Kinase ◽  
Negative Regulator ◽  
Apoptotic Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Altered Expression ◽  
Proinflammatory Mediators ◽  
Downstream Effector ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.

Sign in / Sign up

Export Citation Format

Share Document