Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.     

Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.     

A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.     

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.

F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.

     

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high P_K⁺/P_Na⁺ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K⁺ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.Gliomas are the most common primary tumors of the central nervous system. These tumors arise either from astrocytes or their progenitor cells (1). Gliomas are divided into four grades based on the degree of malignancy. Glioblastoma multiforme (GBM),² Grade IV, is the most frequently occurring, most invasive, and has the worst prognostic outcome with a median survival of approximately one year from diagnosis (2).We have previously reported the presence of an amiloride-sensitive current in glioblastoma cells that is not seen in normal astrocytes or low grade gliomas (3). Amiloride is a potassium sparing diuretic that inhibits sodium channels composed of subunits from the epithelial sodium channel (ENaC)/Degenerin (Deg) family. Amiloride-sensitive Na⁺ channels are essential for the regulation of Na⁺ transport into cells and tissues throughout the body. These channels are found in all body tissues; from epithelia, endothelia, osteoblasts, keratinocytes, taste cells, lymphocytes, and brain (4). Apart from the ENaCs, the ENaC/Deg family also includes acid-sensing ion channels (ASICs) which have been found predominantly in neurons (4–6). Primary malfunctions of ENaC/Deg family members underlie or are involved in the pathophysiology of several human diseases such as salt-sensitive hypertension (7, 8), pseudohypoaldosteronism type I (7), cystic fibrosis (9), chronic airway diseases (10, 11), and flu (12).The ENaC/Deg family subunits share the same structural topology. They all have short intracellular N and C termini, two transmembrane spanning domains, and a large extracellular cysteine-rich loop (4, 5). There are five ENaC subunits termed α, β, γ, δ, and ϵ. Functional ion channels arise from a multimeric assembly of these subunits. The prototypical ENaC channel of the collecting duct principal cell is thought to be αβγENaC (13, 14). The α-ENaC subunit appears to be the core conducting element, whereas the β- and γ-ENaC subunits are associated with trafficking and insertion of the channel in the cell membrane (13, 15, 16). ASICs are homologous to ENaCs and are most prevalently expressed in the brain and nervous system (17–19), although they are also found in the retina (20–22), testes (23), pituitary gland (24), lung epithelia (22), and bone and cartilage (25). Four ASIC genes have been identified so far, ASIC1–4. Of these, ASIC1–3 has multiple splice variants (19, 22). The crystal structure of chicken ASIC1 has revealed it to be a homotrimer (26). ASICs differ from their ENaC counterparts in that they are transiently activated by extracellular acid (19) and are much less sensitive to inhibition by amiloride (27, 28). Also ASIC1 is inhibited with high affinity by psalmotoxin 1 (PcTX-1), a 40-amino acid peptide found in the venom of the West Indies tarantula, Psalmopoeus Cambridgei (29). ASICs, because they are activated by acidic pH, have been suggested to play a role in chemical pain associated with increased tissue acidification as occurs in ischemia (30, 31). They have also been implicated in touch sensation (32), taste (33), fear-conditioning (6), and learning and memory (34).Our laboratory has proposed that ENaC/Deg channels underlie the basally activated cation current measured in high grade glioma cells (3). We hypothesize that the channels forming this current pathway are composed of a mixture of ASIC and ENaC subunits. RNA profiling of a large number of GBM-derived cell lines and freshly resected tumors have revealed the presence of a myriad of ASIC/ENaC components (3). The basally active current seen in GBM cells can be significantly reduced by amiloride or benzamil (a higher affinity amiloride analog), both of which are inhibitors of the ENaC/Deg family of ion channels (3). PcTX1, a selective ASIC1 blocker, also effectively abolishes the basally active GBM current (35). We have previously shown that ENaC and ASIC subunits can form cross-clade interactions in a heterologous expression system (36). This study aims to probe the composition of the novel ENaC/Deg heteromer in a glioma cell line, D54-MG. Our study postulates that a change in GBM cell electrophysiological properties after subunit knockdown would be indicative of that subunit being a part of the GBM channel. We have sequentially knocked down different ENaC/Deg subunits from the D54-MG glioma cells and measured amiloride-sensitive whole cell current using patch clamp. We found that knocking down various ENaC/Deg subunits significantly reduced the whole cell patch clamp current in glioma cells and changed the resting Na⁺/K⁺ permeability of the these cells. After subunit knockdown, glioma cells showed a reduced cell migration as compared with control cells, consistent with our hypothesis that ENaC/Deg subunits play an important role in glioma cell pathophysiology.     

The CFTR and ENaC debate: how important is ENaC in CF lung disease?

Cystic fibrosis (CF) is caused by the loss of the cystic fibrosis transmembrane conductance regulator (CFTR) function and results in a respiratory phenotype that is characterized by dehydrated mucus and bacterial infections that affect CF patients throughout their lives. Much of the morbidity and mortality in CF results from a failure to clear bacteria from the lungs. What causes the defect in the bacterial clearance in the CF lung has been the subject of an ongoing debate. Here we discuss the arguments for and against the role of the epithelial sodium channel, ENaC, in the development of CF lung disease.