Karoly Varga

Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o– airway epithelial monolayers

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP‐regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, ΔF508, results in endoplasmic reticulum‐associated degradation (ERAD) of CFTR. ΔF508 CFTR has been described as a temperature‐sensitive mutation that can be rescued following growth at 27°C. In order to study the processing and function of wild‐type and rescued ΔF508 CFTR at the cell surface under non‐polarized and polarized conditions, we developed stable cell lines expressing ΔF508 or wild‐type CFTR. CFBE41o– is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non‐polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of ΔF508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27°C. However, stimulators of wild‐type CFTR such as forskolin, β2‐adrenergic or A2B‐adenosine receptor agonists failed to activate rescued ΔF508 CFTR in CFBE41o– monolayers. Rescued ΔF508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued ΔF508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl– transport. These results indicate that ΔF508 CFTR, when rescued in CFBE41o– human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild‐type CFTR. Furthermore, the differences in rescue and activation of ΔF508 CFTR in the two cell lines suggest that cell‐type specific differences in ΔF508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the ΔF508 defect.

Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, ∼75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are ∼4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.

Enhanced cell-surface stability of rescued DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones.

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR DeltaF508, and analysed its cell-surface trafficking after rescue [rDeltaF508 (rescued DeltaF508) CFTR]. We show that rDeltaF508 CFTR endocytosis is 6-fold more rapid (approximately 30% per 2.5 min) than WT (wild-type, approximately 5% per 2.5 min) CFTR at 37 degrees C in polarized airway epithelial cells (CFBE41o-). We also investigated rDeltaF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 degrees C) and following treatment with pharmacological chaperones. At low temperature, rDeltaF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rDeltaF508 CFTR is TS. Furthermore, rDeltaF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 degrees C to >8 h at 27 degrees C. Pharmacological chaperone treatment at 37 degrees C corrected the rDeltaF508 CFTR internalization defect, slowing endocytosis from approximately 30% per 2.5 min to approximately 5% per 2.5 min, and doubled DeltaF508 surface half-life from 2 to 4 h. These effects are DeltaF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing DeltaF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

Plasma Membrane CFTR Regulates RANTES Expression via Its C-Terminal PDZ-Interacting Motif

ABSTRACT Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTRs chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane.

ΔF508 CFTR processing correction and activity in polarized airway and non-airway cell monolayers

We examined the activity of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) stably expressed in polarized cystic fibrosis bronchial epithelial cells (CFBE41o(-)) human airway cells and Fisher Rat Thyroid (FRT) cells following treatment with low temperature and a panel of small molecule correctors of DeltaF508 CFTR misprocessing. Corr-4a increased DeltaF508 CFTR-dependent Cl(-) conductance in both cell types, whereas treatment with VRT-325 or VRT-640 increased activity only in FRT cells. Total currents stimulated by forskolin and genistein demonstrated similar dose/response effects to Corr-4a treatment in each cell type. When examining the relative contribution of forskolin and genistein to total stimulated current, CFBE41o(-) cells had smaller forskolin-stimulated I(sc) following either low temperature or corr-4a treatment (10-30% of the total I(sc) produced by the combination of both CFTR agonists). In contrast, forskolin consistently contributed greater than 40% of total I(sc) in DeltaF508 CFTR-expressing FRT cells corrected with low temperature, and corr-4a treatment preferentially enhanced forskolin dependent currents only in FRT cells (60% of total I(sc)). DeltaF508 CFTR cDNA transcript levels, DeltaF508 CFTR C band levels, or cAMP signaling did not account for the reduced forskolin response in CFBE41o(-) cells. Treatment with non-specific inhibitors of phosphodiesterases (papaverine) or phosphatases (endothall) did not restore DeltaF508 CFTR activation by forskolin in CFBE41o(-) cells, indicating that the Cl(-) transport defect in airway cells is distal to cAMP or its metabolism. The results identify important differences in DeltaF508 CFTR activation in polarizing epithelial models of CF, and have important implications regarding detection of rescued of DeltaF508 CFTR in vivo.

Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999)J. Biol. Chem. 274, 3602–3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr1424-X-X-Ile1427where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells

We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl-) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1-1,000 microM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (I(sc)) from 5.2 +/- 0.8 to 15.0 +/- 2.1 microA/cm(2) (X +/- 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1-10 microM) also increased I(sc) by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased I(sc) in Calu-3 cells pretreated with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of I(sc). These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of I(sc) via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-cGMP (up to 2 mM each) did not increase Cl- secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl- secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl- secretion across airway cells without promoting alveolar edema.

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 ∼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 2× 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.