Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice

In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout ( Cftr ^−/−) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr ^−/− mice (23/25) while only a few Cftr^+/+ mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr ^−/− mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr^−/− and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr ^−/− mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.     

The F508del mutation in cystic fibrosis transmembrane conductance regulator gene impacts bone formation

The F508del mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene is believed to be an independent risk factor for cystic fibrosis-related bone disease. In this study, we evaluated the bone mineral density as well as the histomorphometric parameters of bone formation and bone mass in both F508del-Cftr homozygous mice (F508del Cftr(tm1Eur)) and Cftr(+/+) littermate controls at 6 (prepubertal), 10 (pubertal), and 14 (young adult) weeks of age in both sexes. The bone architecture of F508del Cftr(tm1Eur) and wild-type (WT) littermate mice was evaluated by bone densitometry, microcomputed tomography, and analysis of the dynamic parameters of bone formation. Serum levels of both insulin-like growth factor 1 and osteocalcin also were determined. Reduced bone mineral density, lower femoral bone mass, and altered trabecular bone architecture were observed in F508del Cftr(tm1Eur) mice compared with controls at 6, 10, and 14 weeks of age. A decrease in the bone formation rate in F508del Cftr(tm1Eur) mice was shown compared with control mice, independently of age and sex. In addition, we found lower insulin-like growth factor 1 levels in F508del Cftr(tm1Eur) mice compared with age-matched controls, whereas osteocalcin levels were normal. Severe osteopenia and altered bone architecture were found in young and mature adult F508del Cftr(tm1Eur) mice. Our findings show that the F508del mutation in CFTR impacts trabecular bone mass by reducing bone formation.     

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.     

Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.     

Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54

Certain aminoglycosides are capable of inducing "translational readthrough" of premature termination codons (PTCs). However, toxicity and relative lack of efficacy deter treatment with clinically available aminoglycosides for genetic diseases caused by PTCs, including cystic fibrosis (CF). Using a structure-based approach, the novel aminoglycoside NB54 was developed that exhibits reduced toxicity and enhanced suppression of PTCs in cell-based reporter assays relative to gentamicin. We examined whether NB54 administration rescued CFTR protein and function in clinically relevant CF models. In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). In polarized airway epithelial cells expressing either a CFTR-W1282X or -G542X cDNA, treatment with NB54 increased stimulated short-circuit current (I (SC)) with greater efficiency than gentamicin. NB54 and gentamicin induced comparable increases in forskolin-stimulated I (SC) in primary airway epithelial cells derived from a G542X/F508del CF donor. Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents observed in wild-type (Cftr+/+) mice, comparable to gentamicin. NB54 exhibited reduced cellular toxicity in vitro and was tolerated at higher concentrations than gentamicin in vivo. These results provide evidence that synthetic aminoglycosides are capable of PTC suppression in relevant human CF cells and a CF animal model and support further development of these compounds as a treatment modality for genetic diseases caused by PTCs.     

VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.     

Detection of the ΔF508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology

In the present paper, we applied surface plasmon resonance (SPR) and biosensor technologies for biospecific interaction analysis (BIA) to detect ΔF508 mutation (F508del) of the cystic fibrosis transmembrane regulator (CFTR) gene in both homozygous as well as heterozygous human subjects. The proposed method is divided into three major steps. The first step is the immobilization on a SA5 sensor chip of two biotinylated oligonucleotide probes (one normal, N‐508, and the other mutant, ΔF508) that are able to hybridize to the CFTR gene region involved in F508del mutation. The second step consists of the molecular hybridization between the oligonucleotide probes immobilized on the sensor chips and (1) wild‐type or mutant oligonucleotides, as well as (2) single‐stranded DNA obtained by asymmetric polymerase chain reaction (PCR), performed using genomic DNA from normal individuals and from F508del heterozygous and F508del homozygous patients. The third, and most important, step consists of the evaluation of differential stabilities of DNA/DNA molecular complexes generated after hybridization of normal and ΔF508 probes immobilized on the sensor chips. The results obtained strongly suggest that the proposed procedure employing SPR technology enables a one‐step, nonradioactive protocol for the molecular diagnosis of F508del mutation of the CFTR gene. This approach could be of interest in clinical genetics, as the hybridization step is oftenly required to detect microdeletions present within PCR products. Hum Mutat 13:390–400, 1999. © 1999 Wiley‐Liss, Inc.     

CFTR-deficiency renders mice highly susceptible to cutaneous symptoms during mite infestation

Pruritus, also known as itch, is a sensation that causes a desire to scratch. Prolonged scratching exacerbates skin lesions in several skin diseases such as atopic dermatitis. Here, we identify the cystic fibrosis transmembrane conductance regulator (CFTR/Cftr), an integral membrane protein that mediates transepithelial chloride transport, as a determinant factor in mice for the susceptibility to several cutaneous symptoms during mite infestation. Mice that endogenously express dysfunctional Cftr (Cftr(ΔF508/ΔF508)) show significant increase of scratching behavior and skin fibrosis after mite exposure. These phenotypes were due to the increased expression of nerve growth factor (NGF) that augments the sensitization of peripheral nerve fibers. Moreover, protein gene product 9.5 (PGP9.5)-positive neurites were abundant in the epidermis of mite-infested Cftr(ΔF508/ΔF508) mice. Furthermore, mite-infested Cftr(+/+) mice orally administered with a chloride channel inhibitor glibenclamide had higher scratching count and increased level of NGF than vehicle-treated mice. Consistently, mite extract-exposed primary and transformed human keratinocytes, treated with CFTR inhibitor, had significantly higher level of NGF mRNA compared with vehicle-treated, mite extract-exposed cells. These results reveal that CFTR in keratinocytes plays a critical role for the regulation of peripheral nerve function and pruritus sensation, and suggest that Cftr(ΔF508/ΔF508) mice may serve as a novel mouse model that represents NGF-dependent generation of pruritus.     

Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator

Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe. Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-kappa B and cellular desquamation within minutes of P. aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr. Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P. aeruginosa, we found that human bronchial epithelial cells expressing Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR. Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the Delta F508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles. Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P. aeruginosa infection. Prompt apoptosis of infected epithelial cells may be critical for clearance of P. aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.     

Activation of the unfolded protein response by deltaF508 CFTR

Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and deltaF508 CFTR expressed within the same cell.     

An association study on contrasting cystic fibrosis endophenotypes recognizes <Emphasis Type="Italic">KRT8</Emphasis> but not <Emphasis Type="Italic">KRT18</Emphasis> as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion

Background  

F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.     

Population screening if F508del (ΔF508), the most frequent mutation in the CFTR gene associated with cystic fibrosis in Argentina

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. The disease can be caused by one of the more than 900 different mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. However, the deletion of the phe508‐codon is the most prevalent mutation observed. Our aim was to perform a screening for this mutation (ΔF508, or F508del) in the population of Mendoza, Argentina. For the screening, 1,000 blood samples were obtained from CF asymptomatic individuals and combined into 100 pools each containing 10 different blood samples. Pools containing at least one F508del carrier were detected by heteroduplex formation during the PCR amplification of exon 10. The PCR was designed to introduce a recognition site for a restriction enzyme that confirmed the presence of the deletion F508del in the positive pools. The results with this simple method indicate a frequency of carriers in the Mendoza population of 2.1% (1.3%‐3.2, 95% confidence limits). The observed frequency of carriers is similar to that reported for European populations. Hum Mutat 18:167, 2001. © 2001 Wiley‐Liss, Inc.     

Cystic fibrosis patients with mutation 1949del84 in exon 13 of the CFTR gene have a similar clinical severity as delta F508 homozygotes.

The majority of the identified cystic fibrosis (CF) mutations are very uncommon in the total patient population, making the correlation between the clinical presentation and the molecular alterations difficult. The largest deletion that has been described so far in CF is of 84 bp in exon 13, which corresponds to the regulatory (R) domain of the CF transmembrane conductance regulator (CFTR) protein. We have analysed 340 Spanish CF patients for this deletion, named 1949del84, and found three further compound heterozygous patients for mutations 1949del84 and delta F508, and one for 1949del84 and an unknown mutation. Evaluation of the clinical data in these patients suggests that this in-frame deletion, when associated with delta F508, has a similar disease severity to that of delta F508 homozygous patients.     

Isolated elevated sweat chloride concentrations in the presence of the rare mutation S1455X: an extremely mild form of CFTR dysfunction

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause typical cystic fibrosis (CF) and several milder phenotypes. We report on two asymptomatic sisters who had isolated increased sweat chloride concentrations, and in whom systematic scanning of the whole coding region of the CFTR gene revealed the F508del/S1455X genotype.     

Endogenous surface expression of ΔF508-CFTR mediates cAMP-stimulated Cl(-) current in CFTR(ΔF508/ΔF508) pig thyroid epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is both an anion channel and a regulator of other transport proteins. Mutations in the?CFTR?gene underlie the human disease, cystic fibrosis. The most common?CFTR?mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic?CFTR(ΔF508/ΔF508) pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process reliant on functional CFTR. To assess whether endogenous levels of ΔF508-CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn?CFTR(+/+) (wild-type) and?CFTR(ΔF508/ΔF508) (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (I(sc)) compared. Surface biotinylation studies assessed the surface presentation of ΔF508-CFTR. Baseline?I(sc) levels of both wild-type and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in?CFTR(-/-) (knockout) pThECs. Surprisingly, elevation of cAMP transiently increased?I(sc) to peak levels ～65% of those achieved by wild-type. In contrast, knockout pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508-CFTR expression was ～9% that of wild-type, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ～4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508-CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wild-type and ΔF508-CFTR regulate additional thyroid transporters, and together co-ordinate the overall?I(sc) response.     

Effect of acute saline volume expansion in the anaesthetised DeltaF508 cystic fibrosis mouse

It has been suggested that CFTR Cl(-) channels in the renal inner medullary collecting duct may be involved in mediating increased renal salt excretion during extracellular fluid volume expansion. To investigate this hypothesis, in-vivo clearance experiments were performed comparing wild-type (WT) and DeltaF508-CFTR transgenic mice (cftr (tm2Cam)). Control animals were given a 0.1-ml bolus of 0.9% saline, followed by I.V. infusion at 0.3 ml x h(-1). Volume expansion was applied by infusing a 1-ml bolus of 0.9% saline followed by infusion at 0.6 ml x h(-1). No significant differences in renal NaCl handling between WT mice ( C(Na)=1.2 +/- 0.3 microl x min(-1), C(Cl)=4.0 +/- 0.5 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=1.7 +/- 0.5 microl x min(-1), C(Cl)=4.1 +/- 0.8 microl x min(-1)) were observed under control conditions. Volume expansion resulted in large significant increases in NaCl clearance in both WT mice ( C(Na)=7.0 +/- 0.9 microl x min(-1), C(Cl)=12.0 +/- 0.6 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=7.2 +/- 1.6 microl x min(-1), C(Cl)=11.0 +/- 2.2 microl x min(-1)). However, there was no significant difference between WT and DeltaF508-CFTR mice. In conclusion, renal NaCl excretion is not significantly different under basal conditions and during saline volume expansion in DeltaF508-CFTR mice. The data suggest that CFTR is not a physiologically important mediator of volume natriuresis.     

Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency

Sebro R, Levy H, Schneck K, Dimmock D, Raby BA, Cannon CL, Broeckel U, Risch NJ. Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency. Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.