Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.     

Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel in epithelial cells; recently, we identified it in mast cells. Previous work that we confirmed showed that interferon gamma (IFNgamma) down-regulated CFTR expression in epithelial cells (T84), but by contrast, we found that IFNgamma up-regulated CFTR mRNA and protein expression in rat and human mast cells. IFNgamma up-regulation of CFTR in mast cells was inhibited by p38 and extracellular signal-regulated kinase (ERK) kinase inhibitors but not a Janus tyrosine kinase (JAK)2 inhibitor, whereas in T84 cells IFNgamma-mediated down-regulation of CFTR was JAK2-dependent and ERK- and p38-independent. Furthermore, IFNgamma down-regulation of CFTR in T84 epithelial cells was STAT1-dependent, but up-regulation of CFTR in mast cells was STAT1-independent. Thus, differential regulatory pathways of CFTR expression in mast cells and epithelial cells exist that depend upon either p38/ERK or JAK/STAT pathways, respectively. Surprisingly, IFNgamma treatment of mast cells inhibited Cl(-) efflux, in contrast to up-regulation of CFTR/mRNA and protein expression. However, down-regulation of Cl(-) flux correlated with IFNgamma-mediated inhibition of mediator secretion. This and other work suggests that the effect of IFNgamma on CFTR expression in mast cells is important for their function.     

Estrogen inhibition of cystic fibrosis transmembrane conductance regulator-mediated chloride secretion

Cystic fibrosis (CF) is an autosomal genetic disease associated with impaired epithelial ion transport. Mutations in the CF gene alter the primary sequence of the CF transmembrane conductance regulator (CFTR). Several therapeutic modalities have been proposed for CF patients, including the phytoestrogen genistein. Experiments were completed in cellular and subcellular systems to evaluate the impact of naturally occurring and synthetic estrogens on epithelial ion transport, and specifically on the CF protein CFTR. 17beta-Estradiol, a naturally occurring estrogen, caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers with a K(i) of 8 microM. In addition, 17alpha-estradiol, a stereoisomer that fails to bind and activate nuclear estrogen receptors was equipotent with 17beta-estradiol, arguing against a genomic-mediated mechanism of action. Synthetic estrogens, including diethylstilbesterol and the antiestrogen tamoxifen likewise inhibited forskolin-stimulated ion transport. Aldosterone, dexamethasone, and cholesterol were without effect at the highest concentrations tested (>/=1 mM). Studies indicated that diethylstilbesterol and other synthetic estrogens that inhibited anion secretion in intact monolayers likewise inhibited CFTR chloride channel activity with similar concentration dependencies in excised membrane patches. Experiments with radioactive photoactivatable estrogen derivatives demonstrated that these compounds bind directly to CFTR expressed in insect cells. Taken together, the data suggest that estrogens can interact directly with CFTR to alter anion transport.     

A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report on a novel nonsense mutation that leads to exon skipping and the activation of a cryptic exon. Screening of genomic DNA from 700 German patients with CF uncovered four cases with the nonsense mutation E92X, a G-->T transversion that creates a termination codon and affects the first base of exon 4 of the CFTR gene. Lymphocyte RNA of two CF patients heterozygous for E92X was found to contain the wild type sequence and a differentially spliced isoform lacking exon 4. In RNA derived from nasal epithelial cells of E92X patients, a third fragment of longer size was observed. Sequencing revealed the presence of E92X and an additional 183-bp fragment, inserted between exons 3 and 4. The 183-bp sequence was mapped to intron 3 of the CFTR gene. It is flanked by acceptor and donor splice sites. We conclude that the 183-bp fragment in intron 3 is a cryptic CFTR exon that can be activated in epithelial cells by the presence of the E92X mutation. E92X abolishes correctly spliced CFTR mRNA and leads to severe cystic fibrosis.     

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.     

急性心肌梗死后囊性纤维化跨膜转运调节体氯通道基因表达的变化

目的：研究猪急性心肌梗死（AMI）后囊性纤维化跨膜转运调节体（CFTR）氯通道基因表达的变化，探讨急性心肌梗死后早期室性心律失常发生的分子机制。 方法：通过结扎猪左前降支远端1 /3-1 /2处2h然后再灌注建立AMI模型，同时设立相应的假手术(SH)组。术后24h取左心室梗死区、边缘区和正常区内层（Endo）、中层（Mid）和外层（Epi）心肌（SH组取对应位置心肌），应用逆转录－聚合酶链反应(RT－PCR)半定量分析CFTR氯通道基因表达的改变。 结果： 与SH组相比，AMI组CFTR mRNA表达在梗死区三层心肌中均明显下降（P<0.05），在边缘区三层心肌中均明显上升（P<0.05），在正常区三层心肌中均没有显著性改变（P>0.05）。AMI组CFTR mRNA表达在梗死区和边缘区的三层心肌之间以及在梗死区、边缘区和正常区同一层心肌之间有显著性差异（P<0.05）。 结论：AMI后CFTR氯通道基因表达在梗死区、边缘区和正常区心肌之间以及局部心肌三层间呈不均一性改变，可能是AMI后室性心律失常发生的分子生物学基础之一。     

High-efficiency transfer of cystic fibrosis transmembrane conductance regulator cDNA into cystic fibrosis airway cells in culture using lactosylated polylysine as a vector

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.     

Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer

The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.     

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.     

Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer.

In vitro and in vivo studies have demonstrated that gene transfer of the CFTR (cystic fibrosis transmembrane conductance regulator) cDNA into human respiratory cells through nonviral vectors can occur safely and can be done repeatedly. Although functional evaluation of CFTR in cystic fibrosis (CF) patients enrolled in phase I clinical trials using cationic liposomes has shown a partial correction of nasal potential difference, a biological assay indicating a therapeutic relevance of CFTR gene transfer is still missing. Our aims were to study the induction of killing activity toward Pseudomonas aeruginosa (PA) in CF cells by cationic vector-mediated CFTR gene transfer and to use this assay as a therapeutic end point. Luciferase expression and GFP FACS analysis were used to evaluate the optimal vector and the efficiency of gene transfer into non-CF human respiratory cells growing from nasal polyp explants at the air-liquid interface. To prove that transgenic CFTR was expressed in CF cell cultures under the same experimental conditions, a specific RT-PCR was performed. Challenge of the outgrowths with a known amount of PA showed a bacterial clearance activity by non-CF respiratory cells, while in the case of CF cells it even resulted in bacterial growth. Cationic vector-mediated CFTR cDNA determined the recovery of bacterial clearance activity only under those conditions yielding 5% or more of GFP-positive cells. The results shown in this study might be helpful in considering cationic vectors as therapeutic nonviral vectors for transferring CFTR into human CF respiratory cells, as well as for restoring the bacterial killing activity defective in cystic fibrosis.     

Cystic fibrosis transmembrane conductance regulator (CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes

Tumour recurrence following chemotherapy remains a major obstacle to the cure of many cancers. This is exemplified by small-cell lung cancer (SCLC). Host-tumour interactions are central to tumour survival and proliferation. We hypothesized that a factor(s) within the local environment of SCLC cells could provide a survival signal or block a death signal, thereby accounting for the protection of SCLC cells from chemotherapy-induced apoptosis. Here we review recent work undertaken in our laboratory addressing this issue. We have shown that, in vivo, SCLC cells are surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites which contains, among other proteins, fibronectin, laminin and collagen IV. Furthermore, adhesion of SCLC cells to fibronectin, laminin and collagen IV through beta1 integrins enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic agents, including etoposide, cis-platinum and adriamycin. Adhesion to ECM proteins stimulated protein tyrosine kinase (PTK) activity in both untreated and etoposide-treated cells. This effect could be completely blocked by a selective PTK inhibitor or by a function-blocking beta1 integrin antibody. PTK activation was found to block chemotherapy-induced activation of the death protease caspase-3 and, hence, apoptosis. Adhesion to ECM or treatment with a PTK inhibitor did not affect etoposide inhibition of topoisomerase II. Thus adhesion to ECM through beta1 integrins protects SCLC cells from chemotherapy-induced caspase-3 activation and apoptosis by activating PTK signalling downstream of DNA damage. Survival of tumour cells attached to ECM within this microenvironment could explain the local recurrence of SCLC and other tumours that is often seen clinically after chemotherapy.     

Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization.

The fetal pulmonary epithelium secretes fluid. Cl transport is presumed to provide the driving force for net fluid secretion, although the cellular mechanisms have not been well identified in the fetus. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and nucleoside triphosphate-regulated Cl channel; mutations in CFTR cause cystic fibrosis. We hypothesized that if CFTR is involved in fetal lung fluid transport, the fetal pulmonary epithelium should express CFTR mRNA. We used the technique of in situ hybridization with 3H-anti-sense and, as a control, 3H-sense CFTR cRNA probes to localize CFTR mRNA in human fetal lung tissue and cultured lung explants and determine when in gestation it is expressed. Epithelial cells of both first and second trimester lung tissues expressed CFTR mRNA. A decreasing gradient of CFTR mRNA expression was present from the proximal to the distal pulmonary epithelium. Cultured second trimester lung tissue explants expressed more CFTR mRNA than the uncultured starting tissue, suggesting CFTR gene expression increased during the five days in culture. Furthermore, alveolar type II cells in cultured explants expressed CFTR mRNA, suggesting that these cells are Cl-secretory and may be involved in lung fluid transport. These data confirm that CFTR mRNA is expressed in the human fetal pulmonary epithelium, consistent with the Cl-secretory properties of the fetal lung.     

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether deltaF508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells. We have found that, 36Cl- efflux can be stimulated 19-61% above baseline by beta-adrenoreceptor agonists and cGI-phosphodiesterase inhibitors in transformed nasal polyp (CF-T43) cells homozygous for the deltaF508 mutation. The increase in 36Cl- permeability is diminished by protein kinase A inhibitors and is not mediated by an increase in intracellular calcium concentrations. Preincubation of CF-T43 cells with CFTR anti-sense oligonucleotides prevented an increase in 36Cl- efflux in response to beta-agonist and phosphodiesterase inhibitor. Primary cells isolated from CF nasal polyps gave similar results. These data indicate that endogenous levels of deltaF508 protein can be stimulated to increase 36Cl- permeability in airway epithelial cells.     

The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways.

Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl- conductance, are characterized by an enhanced Na+ conductance. In this study we have examined the Na+ conductance in human respiratory tract by measuring transepithelial voltage and resistance (Vte, Rte) and by assessing membrane voltages (Vm) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 micromol/liter) equivalent short circuit current (Isc = Vte/Rte) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 micromol/liter) a significant depolarization of Vm corresponding to the cAMP-dependent activation of a Cl- conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N-methyl-D-glucamine on Vm were attenuated in the presence of forskolin. Also the amiloride-inhibitable Isc was significantly reduced by forskolin (1 micromol/liter) and isobutylmethylxanthine (IBMX; 100 micromol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na+ channels in human airways. This downregulation of epithelial Na+ channels is absent in CF airways, leading to hyperabsorption and to the characteristic increase in mucus viscosity.     

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.     

Restoration of chloride efflux by azithromycin in airway epithelial cells of cystic fibrosis patients

Azithromycin (AZM) has shown promising anti-inflammatory properties in chronic obstructive pulmonary diseases, and clinical studies have presented an improvement in the respiratory condition of cystic fibrosis (CF) patients. The aim of this study was to investigate, in human airway cells, the mechanism by which AZM has beneficial effects in CF. We demonstrated that AZM did not have any anti-inflammatory effect on CF airway cells but restored Cl(-) efflux.     

Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.     

Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.