Na+/H+ Exchanger Regulatory Factor Isoform 1 Overexpression Modulates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Activity in Human Airway 16HBE14o- Cells and Rescues ΔF508 CFTR Functional Expression in Cystic Fibrosis Cells*

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na+/H+ exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous ΔF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent ΔF508 CFBE41o- and ΔF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

The cystic fibrosis transmembrane conductance regulator (CFTR) 3 protein is responsible for the cAMP/PKA-regulated chloride conductance in airway and intestinal epithelia and in exocrine glands. The most common mutation of the gene associated with cystic fibrosis (CF) causes deletion of phenylalanine at residue 508 (⌬F508). This mutation results in the synthesis of a functional but improperly folded CFTR protein that is targeted for degradation mainly via the ubiquitin-proteasome pathway in the ER (1) and, in some tissues, only a negligible amount of ⌬F508 CFTR can reach the plasma membrane of CF cells (2) and transport chloride (3).
A sizable fraction of newly synthesized wild-type (wt) CFTR is also rapidly degraded without ever reaching the plasma membrane, and only 20 -30% of the newly synthesized wt CFTR protein, after passing the ER quality control, is exported from the Golgi to the apical membrane as fully glycosylated CFTR (4). Once delivered to the plasma membrane, CFTR is subjected to rapid internalization to a pool of sub-apical vesicles that can be either recycled to the plasma membrane or delivered to lysosomes for degradation. It has been observed that activation of PKA, in addition to inducing CFTR channel activity, also induces the translocation of CFTR from the sub-apical compartment to the plasma membrane (5)(6)(7). However, this process seems to be tissue-specific, because in some cell systems PKA activation is able to regulate only the activity of CFTR channels already resident in the plasma membrane (8 -10).
PDZ domain proteins have been proposed to be involved in regulation of CFTR localization and activity via their organization of multiprotein complexes at the plasma membrane (11) and regulating, in this way, various functions. CFTR interacts with several PDZ domain proteins such as NHERF, CAP70, and CAL via its C-terminal PDZ-binding motif (12)(13)(14). The Na ϩ /H ϩ exchanger regulatory factor (NHERF) is a 50-kDa membrane protein initially identified as the cofactor required for cAMP inhibition of NHE3 (15), but it is also known to interact with a wide variety of channels, transporters, and receptors (16). The interaction of NHERF with the C terminus PDZ target domain of CFTR (12) has been proposed to have a central role both in stabilizing CFTR at the apical membrane of airway epithelial cells and in the regulation of the CFTR trafficking to the apical membrane, because abrogating CFTR binding to NHERF eliminates both the polarized expression of CFTR on the apical membrane and vectorial chloride transport (17,18).
In addition to PDZ domain interactions, NHERF interacts via its ERM binding domain with ezrin, which is known to act as a PKA anchoring protein and to associate with the actin cytoskeleton (19). This interaction between NHERF, ezrin, and PKA is hypothesized to be essential not only to regulate CFTR activity but also to anchor CFTR to the cytoskeleton (18), to stabilize CFTR protein at the cell surface (20) and to increase the efficiency by which kinases and phosphatases control channel activity (21,22). Cheng et al. (13) suggested that CAL (CFTR-associated ligand), a PDZ-binding protein that interacts with CFTR in the post-ER secretory pathway, may pass CFTR to NHERF in sub-apical vesicles and, therefore, CFTR regulation in the plasma membrane could be the result of a competition between these PDZ proteins for binding with CFTR.
To learn more about the role of NHERF in regulating CFTR in polarized airway cells, we monitored the activity and trafficking of CFTR in two human bronchial cell lines: 16HBE14oϪ cells derived from a normal subject and expressing wild-type CFTR and CFBE41oϪ cells derived from CF individuals homozygous for the ⌬F508 mutation of CFTR. The 16HBE14oϪ cell monolayers provide a well polarized airway epithelial cell culture system exhibiting vectorial ion transport (23) and express NHERF1 primarily on the apical membrane where it functions as a scaffolding protein organizing apical membrane proteins into regulatory complexes (24). We report here that both normal and CF cells express both NHERF1 and NHERF2, but it is NHERF1 that follows CFTR expression patterns. Both of the PDZ1 and PDZ2 domains and the ERM domain of NHERF1 are involved in the polarized expression of CFTR and in the regulation of CFTR-dependent chloride efflux. Further, targeted NHERF1 overexpression stimulates CFTR-dependent chloride efflux by increasing apical CFTR expression in normal 16HBE14oϪ cells. Importantly, in the CF cell lines, CFBE41oϪ and CFT1-C2, targeted NHERF1 overexpression induced the redistribution of CFTR from the cytoplasm to the plasma membrane and rescues CFTR activity.

EXPERIMENTAL PROCEDURES
Cell Culture-Experiments were performed with a series of human tracheobronchial epithelial cell lines: the normal, 16HBE14oϪ, and the CF homozygous for the ⌬F508 allele (⌬F508/⌬F508), CFBE41oϪ, comprised the generous gift of Prof. D. Gruenert (California Pacific Medical Center Research Institute, University of California at San Francisco), whereas the CF cell line, CFT1-C2, isolated from a ⌬F508 CF patient, was the generous gift of Prof. J. R. Yankaskas (Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC). 16HBE14oϪ and CFBE41oϪ were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin at 37°C under 5% CO 2 . CFT1-C2 cells were cultured with serum-free Ham's F-12 supplemented with 10 g/ml insulin, 3.7 g/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, 30 nM triiodothyronine, 1 M hydrocortisone, 5 g/ml transferrin, and 10 ng/ml cholera toxin (Sigma) (25). All cell lines were routinely grown on tissue culture plastic flasks coated with an extracellular matrix containing fibronectin/vitrogen/bovine serum albumin (26). For experiments of chloride efflux, confocal immunofluorescence analysis, Western blot analysis and biotinylation, cells were seeded on 0.4-m pore size PET filter inserts (Falcon BD Biosciences Labware) coated with the same extracellular matrix.
Transfection of NHERF1 and NHERF2 cDNAs-At 70 -80% confluence, cells were transiently transfected with wild-type mouse NHERF1 cDNA or cDNAs mutated in the different domains and inserted into the pcDNA3.1/Hygroϩ vector. HRF1A and HRF2A are cDNAs encoding NHERF1 in which alanine substitutions GAGA in the core peptidebinding sequence, GYGF, in PDZ1 (HRF1A) and PDZ2 (HRF2A) domain inactivate the individual PDZ domain; ⌬ERM is a cDNA encoding NHERF1 truncated of the last 30 amino acids. All of these clones behave as dominant mutants over the endogenous protein. Another plasmid construct used was human, wild-type NHERF2 inserted into the pcDNA3.1/His-C (gift of Dr. Pann Ghill Suh). The cells were transiently transfected using Escort IV reagent (Sigma) according to the manufacturer's protocol, and the experiments were conducted 48 h later.
Fluorescence Measurements of Apical Chloride Efflux-Chloride efflux was measured using the Cl Ϫ -sensitive dye MQAE (26). Confluent cell monolayers grown on permeable filters were loaded overnight in culture medium containing 5 mM MQAE at 37°C in a CO 2 incubator and then inserted into a perfusion chamber that allowed independent perfusion of apical and basolateral cell surfaces. Fluorescence was recorded with a Cary Eclipse Varian spectrofluorometer using 360 nm (bandwidth 10 nm) as excitation wavelength and 450 nm (bandwidth 10 nm) as emission wavelength. To measure chloride efflux rate across the apical membrane, the apical perfusion medium was changed to a medium in which chloride was substituted with iso-osmotic nitrate. All experiments were performed at 37°C in HEPES-buffered bicarbonatefree media (Cl Ϫ medium (in millimolar): NaCl 135, KCl 3, CaCl 2 1.8, MgSO 4 0.8, HEPES 20, KH 2 PO 4 1, glucose 11, and Cl Ϫ free-medium: NaNO 3 135, KNO 3 3, MgSO 4 0.8, KH 2 PO 4 1, HEPES 20, Ca(NO 3 ) 2 5, glucose 11). At the end of each experiment a calibration procedure was performed as previously described (26). The rates of chloride efflux were calculated by linear regression analysis of the first 30 points taken at 4 s intervals while the change of fluorescence was still linear. In all reported experiments, the basolateral side of the monolayer was treated with bumetanide (5 M) for 5 min before stimulation to avoid the possibility that the observed increase of chloride efflux could be due to the stimulation of basolateral Na ϩ /K ϩ /2Cl Ϫ .
Detection of CFTR-dependent Chloride Efflux-16HBE14oϪ polarized monolayers exhibited a basal chloride efflux under baseline conditions when chloride was replaced by apical nitrate (0.030 Ϯ 0.003 ⌬(F/ F 0 )/min, n ϭ 15). Stimulation of PKA by addition of FSK plus IBMX (first traces of the typical experiments in supplemental Fig. 1S) significantly increased this apical chloride efflux (0.052 Ϯ 0.005 ⌬(F/F 0 )/min, n ϭ 15, p Ͻ 0.0001). It is important to note that this PKA-dependent increase was specific for apical chloride efflux, because the chloride efflux induced by basolateral nitrate perfusion was not altered by FSK plus IBMX (0.032 Ϯ 0.02 versus 0.031 Ϯ 0.004 ⌬(F/F 0 )/min before and after FSK plus IBMX treatment, respectively, n ϭ 4, n.s.). The typical traces in supplemental Fig. 1S, show that addition of the CFTR inhibitors, glibenclamide (Fig. 1S, A, 100 M) (27) or the highly specific CFTR inh -172 ( Fig. 1S, C, 5 M) (28,29) to the apical perfusion fluids, before (5 min) and during the next FSK plus IBMX stimulation, inhibited this PKA-dependent increase to basal levels. Fig. 1S (B and D) shows the summary of these experiments and, in the histogram, the empty bar represents CFTR-dependent chloride efflux calculated as the difference in alterations of FSK plus IBMX stimulated fluorescence in the absence (light gray bar) and presence (dark bar) of the above mentioned CFTR inhibitors. Because the CFTR-dependent Cl Ϫ effluxes calculated with the two inhibitors (empty bars) were not significantly different from each other (0.024 Ϯ 0.002, n ϭ 15, versus 0.026 Ϯ 0.007, n ϭ 6, ⌬(F/F 0 )/ min with glibenclamide and CFTR inh -172, respectively, n.s.), we used glibenclamide in all the rest of the experiments and define CFTR-dependent chloride efflux as the difference between the rate of FSK plus IBMX-stimulated chloride efflux before and after apical glibenclamide treatment (presented as the empty bar in all subsequent figures).
Protein Extraction and Western Blotting-Confluent 16HBE14oϪ and CFBE41oϪ monolayers grown on coated permeable filters, were washed with PBS, lysed in lysis buffer A (NaCl 110 mM, Tris 50 mM, Triton X-100 0.5%, and Igepal CA-630 0.5%, pH 8, with added protease inhibitor mixture), sonicated for 10 s, and centrifuged for 10 min (16,000 ϫ g), and then the pellet was discarded. Supernatant protein concentration was measured by the method of Bradford (30), and an aliquot of 30 g of protein was diluted in Laemmli buffer, heated at 100°C for 5 min, and separated by 4 -12% SDS-PAGE Criterion XT precast gel (Bio-Rad). The separated proteins were transferred to Immobilon P (Millipore) in a Trans-Blot semidry electrophoretic transfer cell (Amersham Biosciences) for immunoblotting. The primary antibodies used were anti-hCFTR monoclonal antibody against the C terminus (R&D Systems, MAB25031, dilution 1:500), anti-hNHERF1 monoclonal antibody (BD Transduction Laboratories, dilution 1:500), and anti-hNHERF2 polyclonal antibody (Alpha Diagnostic International, dilution 1:1000). The secondary antibodies were anti-mouse IgG for monoclonal antibodies and anti-rabbit IgG for polyclonal antibody (Sigma). Immunocomplexes were detected with ECL plus reagent (Amersham Biosciences) and densitometric quantification and image processing were carried out using Adobe Photoshop and the Image software package (version 1.61, National Institutes of Health, Bethesda, MD).
Cell Fractionation-Fractionation was performed as described by Sun et al. (31). Confluent 16HBE14oϪ and CFBE41oϪ cells, cultured on coated permeable filters, were scraped into lysis buffer B (Tris-HCl, 10 mM (pH 7.4), NaCl, 50 mM, EDTA, 1 mM, with protease inhibitor mixture), homogenized in a 5-ml syringe with a 0.8 ϫ 40-mm needle, and an aliquot of total homogenate was collected. Postnuclear supernatants were obtained by centrifugation (900 ϫ g for 15 min) at 4°C. Another centrifugation (20,000 ϫ g for 20 min) was performed, the pellet was discarded, and the supernatant was centrifuged again at 100,000 ϫ g for 60 min at 4°C to obtain cytosolic and membrane fractions. Cytosolic proteins were prepared by concentrating the supernatants through Centricon Centrifugal Filter Devices YM-10, M r 10,000 cut-off (Millipore), whereas the membrane fraction was obtained by resuspending the pellet in resuspension buffer (HEPES, 50 mM (pH 7.4), NaCl, 150 mM, EDTA, 1 mM, 1% Nonidet P-40, 10% glycerol). 30 g of protein from total lysate, membrane, and cytosolic fractions were diluted in Laemmli buffer and resolved by 4 -12% SDS-PAGE Criterion XT precast gel (Bio-Rad).
Biotinylation of Apical Membrane Proteins-16HBE14oϪ and CFBE41oϪ cells, grown on coated permeable filters, were transfected with the vector containing wt NHERF1, PDZ domain-mutated or ERM domain-deleted NHERF1 cDNA constructs, or the empty vector. 48 h later the monolayers were washed with PBS and incubated with 2 mg/ml sulfo-NHS-biotin (Sigma) in PBS for 30 min at 4°C. All further steps were performed in a cold room. Free sulfo-NHS-biotin was removed by washing cells twice at 4°C with 0.1 M glycine in PBS and then with PBS. Cells were lysed in lysis buffer A, sonicated, and centrifuged, and the pellet was discarded. Volumes of supernatant, containing equal amounts of protein, were incubated overnight at 4°C with gentle mixing with the same amount of streptavidin-agarose beads (Pierce) (50 l of streptavidin/mg of biotin). Streptavidin-bound complexes were pelleted (16,000 ϫ g), and after two washes with buffer lysis, biotinylated proteins were eluted in Laemmli buffer. The eluted proteins were subjected to SDS-PAGE and Western blotting as described above.
Immunofluorescence Analysis-10 5 cells were seeded on round coated glass coverslips and 48 h later washed with PBS, fixed in 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized in 0.1% Triton X-100 in PBS for 10 min. After three more washes in PBS, the cells were blocked in 0.1% gelatin in PBS for 10 min and then incubated in a wet environment with primary antibodies for 1 h; anti-hNHERF1 (dilution 1:50) and anti-hNHERF2 (dilution 1:50). After washing three times with 0.1% gelatin in PBS, the cells were incubated in a wet environment with secondary antibodies for 1 h: goat anti-mouse IgG conjugated to Alexa Fluor 488 (Molecular Probes, dilution 1:1000) or goat anti-rabbit IgG conjugated to Alexa Fluor 568 (Molecular Probes, dilution 1:1000). After three washes in PBS, coverslips were mounted onto slides with Vectashield mounting medium (Vector Laboratories). Fluorescence data were collected with a Nikon Eclipse TE 2000-S with a 40ϫ oil immersion objective and processed by using the Metamorph Imaging System (Meta Imaging 9.1). Deconvolution was performed using Auto Deblur software (AutoQuant Imaging Suite 9.1). Collected images were exported to Adobe Photoshop for subsequent analyses.
Confocal Imaging-For confocal immunofluorescence analysis, cells grown on coated permeable filters were prepared as described above and examined using a Zeiss Axioskop microscope equipped with a laser scanning confocal unit model MRC-1024 containing a 15-milliwatt krypton-argon laser (Bio-Rad). Monolayer confluence was detected by staining monolayers for the tight junction protein, ZO-1 (mouse anti-ZO-1-(Alexa Fluor 488) Zymed Laboratories, dilution 1:50). Specimens were viewed through a Planapo 63ϫ/1.4 oil immersion objective, and images were randomly acquired in the vertical plane (xz) by the Laser Sharp 2000 program (Bio-Rad). The relative distribution of CFTR (Alexa Fluor 568) and NHERF1 (Alexa Fluor 488) was determined in the apical region and in the cytoplasm by semiquantitative analysis of previously acquired images using ImageJ software: a freehand selection (ϳ8 m 2 ) was drawn over the region to be measured, and pixel counts within the selected region were determined. The transition between apical and basolateral regions was identified by staining lateral membranes of the monolayers for the tight junction protein ZO-1. The distribution of CFTR and NHERF1 was calculated using the formula, r ϭ a/c, where a corresponds to pixel counts in the apical region and c corresponds to pixel counts in the cytoplasmic region.
Data Analysis-Data are presented as means Ϯ S.E. for the number of samples indicated (n). Statistical comparisons were made using either unpaired or paired data Student's t test. Differences were considered significant when p Ͻ 0.05.

Expression and Distribution of NHERF1 and NHERF2 in 16HBE14oϪ
and CFBE41oϪ Cells-Studies have provided evidence that CFTR interacting proteins play critical roles in determining its proper expression and function, and NHERF was the first identified CFTR-binding protein (32). To further clarify the mechanism by which the interaction of NHERF with CFTR regulates CFTR expression and activity in human airway epithelial cells, we first analyzed the expression levels and cellular distribution of both NHERF isoforms in the normal human bronchial epithelial cell line, 16HBE14oϪ and in the ⌬F508 homozygous (⌬F508/ ⌬F508) CFBE41oϪ cell line. Western blot analysis performed in the airway cells seeded on coated permeable filters revealed that both the NHERF1 Modulation of CFTR Activity in Airway Cells DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 NHERF1 and NHERF2 isoforms were expressed in both cell lines (Fig.  1A). Therefore, we next examined the cellular distribution of NHERF1 and NHERF2 between the surface and the inside of the cell by indirect immunofluorescence staining with mouse anti-NHERF1 and rabbit anti-NHERF2. Interestingly, both cell lines revealed an intracellular distribution for NHERF2 (Fig. 1B), whereas NHERF1 was localized at the cell surface in 16HBE14oϪ cells and was mainly cytoplasmic in CFBE41oϪ cells (Fig. 1B). This finding that only NHERF1 distribution is different between 16HBE14oϪ and CFBE41oϪ cells directed us to focus on a more complete analysis of the localization of NHERF1 and CFTR in polarized cultures of the two cell lines.
CFTR and NHERF1 Expression and Localization in 16HBE14oϪ and CFBE41oϪ Cell Monolayers-We first determined the relative amount of mature (180 kDa) and immature (160 kDa) CFTR in lysate, cytosol, and total membrane fractions of cell monolayers grown on permeable coated filters ( Fig. 2A). In 16HBE14oϪ cell monolayers (HBE), wt CFTR was found both as a mature (180 kDa) and an immature (160 kDa) band in the lysate and cytosolic fractions and as only the mature band in the total membrane fraction, whereas, in CFBE41oϪ cell monolayers (CF), ⌬F508 CFTR was found mainly as the immature band, although a small amount of mature ⌬F508 CFTR was detectable in the total membrane fraction as has been reported in other CF cells (2). Regarding NHERF1 distribution, in 16HBE14oϪ cells NHERF1 was expressed in both the cytosol and membrane fractions, whereas it was absent in the membrane fraction of CFBE41oϪ cells. Moreover, NHERF1 was less expressed in CFBE41oϪ compared with 16HBE14oϪ cells (Ϫ32.29 Ϯ 6.42%, n ϭ 3, p Ͻ 0.05 and Ϫ30.59 Ϯ 6.93%, n ϭ 3, p Ͻ 0.05 in the lysate and cytosolic fractions, respectively).
As these fractionation data suggest that CFTR and NHERF1 are more associated with the total membrane fraction in normal cells than in CF cells, it was necessary to obtain a more detailed analysis of CFTR and NHERF1 cellular distribution. We next performed confocal microscopy analysis of 16HBE14oϪ and CFBE41oϪ cells grown on permeable sup-port. Fig. 2B demonstrates that in 16HBE14oϪ cells CFTR was distributed close to the apical region, whereas its distribution in CFBE41oϪ cells was primarily cytoplasmic. NHERF1 distribution in these polarized cell monolayers also was primarily associated with the apical membrane region in 16HBE14oϪ cells while being cytoplasmic in CFBE41oϪ cells.
PKA-dependent CFTR Activity in 16HBE14oϪ and CFBE41oϪ Cells-This similarity in the distribution of NHERF1 and CFTR in 16HBE14oϪ and CFBE41oϪ cells directed us to then focus on the analysis of the role of NHERF1 in the regulation of CFTR activity. The rate of chloride efflux after substitution of chloride by nitrate in the apical perfusion medium was measured by the change in fluorescence (⌬(F/ F 0 )/min) of the chloride-sensitive dye, MQAE (see "Experimental Procedures" and supplemental Fig. 1S). Fig. 3 illustrates a typical experiment (A and C) and the summary (B and D) of 15 experiments of chloride efflux in 16HBE14oϪ cell monolayers (A and B) and 10 experiments of chloride efflux in ⌬F508 CFBE41oϪ cell monolayers (C and D). As in other cell types (27,31,32), both 16HBE14oϪ and CFBE41oϪ polarized monolayers exhibited a basal chloride efflux under baseline conditions when chloride was replaced by nitrate that were not significantly different (0.030 Ϯ 0.003, n ϭ 15 versus 0.022 Ϯ 0.002, n ϭ 10, ⌬(F/F 0 )/min in 16HBE14oϪ and CFBE41oϪ, respectively, n.s.). Stimulation of PKA by addition of FSK plus IBMX (first trace of the typical experiment in Fig. 3, A and C, and the first bar in Fig. 3, B and D) significantly increased chloride efflux in 16HBE14oϪ cells (0.052 Ϯ 0.005 ⌬(F/F 0 )/min, n ϭ 15, p Ͻ 0.0001) while having no effect in CFBE41oϪ cells (0.023 Ϯ 0.003 ⌬(F/F 0 )/min, n ϭ 10, n.s.). The addition of the CFTR inhibitor, glibenclamide, before and during the next FSK plus IBMX stimulation (second trace of the typical experiment in Fig. 3, A and C, and the second bar in Fig. 3, B and D) inhibited this increase to basal levels in 16HBE14oϪ cells (0.027 Ϯ 0.004 ⌬(F/F 0 )/min, n ϭ 15, p Ͻ 0.0001) while having no effect in CFBE41oϪ cells (0.020 Ϯ 0.002 ⌬(F/F 0 )/min, n ϭ 10, n.s.). The glibenclamide inhibition was completely reversible as is shown in the third trace of the typical experiment in Fig.  3A in which the chloride efflux stimulated by FSK plus IBMX is identical to the first trace. Similar results were obtained using another specific FIGURE 1. Presence of both NHERF isoforms in cultured human bronchial epithelial cells. A, 30 g of total cell lysate, of 16HBE14oϪ and CFBE41oϪ cells, grown on permeable filters, was analyzed by Western blot analysis using anti-NHERF1 monoclonal antibody or anti-NHERF2 polyclonal antibody as described under "Experimental procedures." Human endothelial cell lysate was used as positive control for NHERF1 and as negative control for NHERF2 in Western blotting experiments. B, NHERF1 and NHERF2 were detected by immunofluorescence in cells grown on coated glass coverslips as described under "Experimental Procedures" using anti-NHERF2 antibody (red) and anti-NHERF1 antibody (green). Scale bars, 10 ϭ m. inhibitor of CFTR, the thiazolidinone CFTR inh -172 (28,29), which has been demonstrated to alter CFTR gating (see supplemental Fig. 1S). CFTR-dependent chloride efflux is defined as the difference between the rate of FSK plus IBMX-stimulated chloride efflux before and after apical glibenclamide treatment (third bar in Fig. 3, B and D) and, as can be seen in those figures, CFTR-dependent chloride efflux in 16HBE14oϪ cells was high (0.024 Ϯ 0.002 ⌬(F/F 0 )/min, n ϭ 15, p Ͻ 0.0001), whereas in CFBE41oϪ cells it was not significantly different from zero (n ϭ 10, n.s.). A similar lack of significant CFTR-dependent chloride efflux in CFBE41oϪ cells were obtained in using the specific inhibitor CFTR inh -172 (5 M) (data not shown).

Role of NHERF1 PDZ Domains in Regulating CFTR Expression and Activity in 16HBE14oϪ
Cells-To learn more about the role and mechanism of NHERF1 and its different binding domains in regulating CFTR expression, we measured apical CFTR expression by surface biotinylation after transfection of permeable filter grown 16HBE14oϪ monolayers with His 6 -tagged wild-type, PDZ domain-mutated, or ERM domaindeleted NHERF1 constructs (33). Immunofluorescence analysis of the transfected monolayers demonstrated that ϳ57 Ϯ 4% (n ϭ 15) of the cells were transfected. Treatment with either the transfection vehicle, Escort IV, or transfection with the empty vector, pcDNA3.1/hygroϩ, did not significantly alter either apical CFTR expression in biotinylation experiments or CFTR-mediated chloride transport (data not shown).
As shown in Fig. 4 (A and B), the transfection of 16HBE14oϪ monolayers with wt NHERF1 induced an increase of biotinylated, apical CFTR compared with non-transfected cells (ϩ46.90 Ϯ 3.99%, n ϭ 4, p Ͻ 0.01). In contrast, overexpression of NHERF2 had no effect on apical CFTR protein expression (Ϫ2.75 Ϯ 8.26%, n ϭ 3, compared with non-transfected cells, n.s.). Transfection with cDNA encoding NHERF1 with mutations in the GYGF core peptide-binding groove sequence to GAGA that inactivates either the PDZ1 or PDZ2 domain (HRF1A and HRF2A, respectively) or with the cDNA encoding ERM domain (⌬ERM) deleted NHERF1, significantly reduced the levels of the apical CFTR band. These results confirm previous studies showing that the deletion of the PDZ interacting domain of CFTR abrogating the interaction between CFTR and NHERF inhibited the polarized expression of CFTR on the apical membrane (17,34). Moreover, the finding that the deletion of ERM domain diminished the apical CFTR expression is in line with the hypothesis that the association of CFTR with the cytoskeleton by the ERM domain could serve as an anchor that determines its specific location at the apical membrane.
We next analyzed the role of NHERF1 and its different binding domains in altering CFTR-dependent chloride secretion (Fig. 5). As with apical CFTR expression, transfection of 16HBE14oϪ monolayers with wt NHERF1 (Fig. 5) significantly potentiated CFTR-dependent chloride secretion, whereas transfection of the cell monolayers with wt NHERF2 had no effect on CFTR-dependent chloride efflux (0.024 Ϯ 0.002, n ϭ 15 versus 0.026 Ϯ 0.006, n ϭ 5, ⌬(F/F 0 )/min, in the nontransfected and NHERF2 transfected monolayers, respectively, n.s.) confirming that only NHERF1 overexpression augments CFTR-mediated Cl Ϫ secretion by increasing the CFTR protein expression in the apical membrane. Transfection of 16HBE14oϪ monolayers with the cDNA encoding NHERF1 with the mutated PDZ1 (HRF1A) or PDZ2 (HRF2A) almost completely abrogated CFTR-dependent chloride  ϭ 10), where CFTR-dependent chloride efflux (empty bar) was calculated as the difference in the F/F 0 ratio per minute (⌬(F/F 0 )/min) in the absence of (light gray bar) and presence of (dark bar) glibenclamide. Each bar represents the mean Ϯ S.E. Statistical comparison was made using paired Student's t test with respect to the chloride efflux stimulated by FSK plus IBMX measured in the absence of glibenclamide. DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 efflux, demonstrating that the interaction of both NHERF1 PDZ domains with the C terminus of CFTR, in addition to being important in the polarized expression of CFTR, are fundamental for the regulation of CFTR activity as observed in other cell lines (35,36). Further, transfection of 16HBE14oϪ monolayers with the cDNA encoding the ⌬ERM deletion of the NHERF1 ERM domain, which interacts with ezrin, inhibited PKA-dependent regulation of CFTR as was expected, because ezrin serves as an anchor for protein kinase A (PKA-anchoring protein) and tethers PKA directly to CFTR for efficient and specific phosphorylation (19). Confirmation for this hypothesis came from experiments in which the preincubation of 16HBE14oϪ cell monolayers with 100 M S-Ht31, which prevents the binding between PKA anchoring proteins andTypeIIregulatorysubunitsofPKA(37),completelyabrogatedCFTRdependent chloride efflux (0.005 Ϯ 0.003 ⌬(F/F 0 )/min, n ϭ 3).

NHERF1 Modulation of CFTR Activity in Airway Cells
Overexpression of NHERF1 Rescues ⌬F508 CFTR Functional Localization in CFBE41oϪ Cells-The increase of both the apical CFTR expression and CFTR-dependent chloride efflux by overexpression of wild-type NHERF1 in 16HBE14oϪ cells suggests the possibility that targeted overexpression of NHERF1 might be able to rescue the activity and expression of CFTR in the ⌬F508 CFBE41oϪ cells. Transfection of CFBE41oϪ monolayers with wt NHERF1 increased total NHERF1 expression by 129 Ϯ 10% (n ϭ 4, p Ͻ 0.001, Fig. 6A) and resulted in a 39 Ϯ 10% (n ϭ 4, p Ͻ 0.05) increase in apical CFTR expression with respect to non-transfected monolayers as measured by apical biotinylation of polarized CFBE41oϪ cell monolayers grown on permeable filters (Fig. 6B). Overexpression of NHERF2 had no effect on apical CFTR protein expression (ϩ4.45 Ϯ 7.38% compared with non-transfected cells, n ϭ 3, n.s.), demonstrating that, as in 16HBE14oϪ cells, in CFBE41oϪ cells only the overexpression of NHERF1 is able to increase ⌬F508 CFTR apical expression. These results were confirmed by confocal analysis of confluent CFBE41oϪ cell monolayers in which it can be observed that after overexpression of wt NHERF1 both ⌬F508 CFTR and NHERF1 are confined predominantly to the apical membrane   Fig. 4. After 48 h the Cl Ϫ transport activity of CFTR was determined, and CFTR-dependent chloride efflux was calculated as described in Fig. 3B. Statistical comparisons were made using unpaired Student's t test with respect to the values obtained in non-transfected monolayers. FIGURE 6. Transfection with cDNA for wild-type NHERF1 increases cell surface CFTR expression in CFBE41o؊ cell monolayers. CFBE41oϪ cells, grown on permeable filters, were transfected with wt NHERF1 or treated with the transfection vehicle, Escort IV, as described under "Experimental procedures." A, total lysates were analyzed using anti-NHERF1 antibody. B, cell-surface membrane proteins were biotinylated and analyzed by Western blot analysis using anti-CFTR antibody as described under "Experimental Procedures." Similar results were obtained in four independent experiments. FIGURE 7. Localization of NHERF1 and CFTR in CFBE41o؊ and CFT1-C2 cells by confocal immunofluorescence analysis. Confocal immunofluorescence microscopy was performed as described under "Experimental Procedures" in CFBE41oϪ and CFT1-C2 cells non-transfected (treated with the transfection vehicle, Escort IV) or transfected with cDNA for wt NHERF1. CFTR was detected by monoclonal antibody (red), and NHERF1 was detected by monoclonal antibody (green) as described in Fig. 2, and all images are in the vertical (xz) plane. Scale bars ϭ 10 m. In CFBE41oϪ cell monolayers, the calculated pixel density ratios (R) of the apical region versus the cytoplasmic region of ⌬F508 CFTR (see "Experimental Procedures") were: 3.57 Ϯ 0.91 (n ϭ 4) and 1.06 Ϯ 0.09 (n ϭ 4, p Ͻ 0.05) and the ratios (R) for NHERF1 expression were 2.80 Ϯ 0.68 (n ϭ 4) and 1.1 Ϯ 0.13 (n ϭ 4, p Ͻ 0.05) in transfected and non-transfected monolayers, respectively. Similarly, in CFT1-C2 cells the ratios for ⌬F508 CFTR were 3.34 Ϯ 0.24 (n ϭ 3) and 0.97 Ϯ 0.08 (n ϭ 4, p Ͻ 0.001), and the ratios for NHERF1 were 2.05 Ϯ 0.039 (n ϭ 4) and 0.98 Ϯ 0.07 (n ϭ 5, p Ͻ 0.02) in transfected and non-transfected cells, respectively. region (Fig. 7), even if we cannot discriminate between apical or subapical areas because we have not specifically stained for the apical membrane. To determine that this behavior is not specific for CFBE41oϪ cells, we overexpressed NHERF1 in another CF airway cell line also having the ⌬F508 CFTR mutation: the human tracheal epithelial cell line, CFT1-C2 (25). We observed that these cells also express endogenous NHERF1 and, as observed above in the CFBE41oϪ cells, targeted NHERF1 overexpression shifted both ⌬F508 CFTR and NHERF1 localization from the cytoplasm to the apical plasma membrane region (Fig. 7).

DISCUSSION
NHERF1 (EBP50) and NHERF2 (E3KARP) were initially proposed as cofactors necessary for the cAMP-dependent regulation of NHE3 (38), and a growing body of data has demonstrated that both NHERF isoforms regulate a variety of transporters, channels, and receptors by facilitating the formation of multiprotein signaling complexes (39). A large body of studies has recently focused on the role of NHERF in regulating CFTR activity. NHERF interacts with CFTR via its two PDZ domains, although with a higher affinity for the PDZ1 domain than for the PDZ2 domain (18,32,35) and, in this way, possibly free the PDZ2 domain to interact with other proteins co-expressed on the same membrane such as ROMK (40) or NHE3 (26,41). In addition to regulating PKA-dependent CFTR activity, NHERF can also influence apical expression of CFTR (17,34) and retention of CFTR in the apical membrane (20).
In many but not all epithelial cells, the expression of the two isoforms of NHERF (NHERF1 and NHERF2) is mutually exclusive. For example, in the terminal bronchioles, NHERF1 co-expressed with ezrin is the only isoform present (42), whereas in another airway cell model, Calu3,  DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 both isoforms are expressed and compete for CFTR binding (31). Here, we observe that both the NHERF isoforms are present in the normal human bronchial epithelial cell line, 16HBE14oϪ, although with a different localization: NHERF1 is present close to the plasma membrane, as previously reported in the same cell line (24), whereas NHERF2 is diffusely distributed in the cytoplasm (Fig. 1). Interestingly, in cells derived from a CF individual homozygous for the ⌬F508 mutation of CFTR, CFBE41oϪ, both NHERF isoforms are diffusely distributed in the cytoplasm (Fig. 1).

NHERF1 Modulation of CFTR Activity in Airway Cells
Importantly, the distribution of CFTR in both 16HBE14oϪ and CFBE41oϪ cells, as detected by confocal immunolocalization, paralleled the distribution of NHERF1: in 16HBE14oϪ-polarized cell monolayers, CFTR and NHERF1 are present predominantly at the apical membrane region, whereas in the CF cells, ⌬F508 CFTR and NHERF1 are almost absent in the plasma membrane region but are diffusely distributed in the cytoplasm (Fig. 2B). As could be expected from the different distribution of CFTR, polarized 16HBE14oϪ monolayers displayed a large CFTR-mediated Cl Ϫ apical secretion, whereas in polarized CFBE41oϪ monolayers the CFTR-mediated Cl Ϫ secretion was completely absent. Comparable data have been reported in cultured normal and CF airway cells (43).
The similarity in the distribution of NHERF1 and CFTR in 16HBE14oϪ and CFBE41oϪ cells directed us to focus on the analysis of the role of NHERF1 in the regulation of CFTR expression and activity. We observed that both PDZ1 and PDZ2 domains of NHERF1 are equally involved in the polarized expression of CFTR and in the regulation of CFTR-dependent chloride efflux. Our data are consistent with the findings demonstrating that PDZ domains direct the polarized apical expression of CFTR (17,20,34,44) and support the role of NHERF in forming apical multiprotein complexes that permit the micro-compartmentalization of signal transduction modules promoting efficient regulation of CFTR (18). Deletion of the ERM domain similarly reduced both apical CFTR expression and the PKA-dependent regulation of CFTR, which could be explained by the fact that NHERF1 has been demonstrated to associate by ERM domain with the cytoskeletal adaptor protein, ezrin, which is also a protein kinase A-anchoring protein, and, in this way, NHERF both compartmentalizes PKA in the vicinity of CFTR protein and regulates apical CFTR activity by promoting CFTR phosphorylation (31). On the contrary overexpression of wt NHERF1 in 16HBE14oϪ cells dramatically increased both the apical CFTR expression and the PKA-dependent regulation of CFTR activity.
All together these findings lead us to hypothesize that, as it has been demonstrated in other polarized cells, in 16HBE14oϪ cells NHERF1 may regulate CFTR-dependent chloride secretion by influencing either the efficiency of CFTR recycling (20) and/or the stability of CFTR in the plasma membrane via anchoring CFTR to the cytoskeleton (45).
The most important finding of the present report comes from the effect of targeted wt NHERF1 overexpression in confluent monolayers of two CF cell lines, CFBE41oϪ and CFT1-C2. NHERF1 overexpression induced (a) a significant redistribution of CFTR from the cytoplasm to the apical region, (b) an increase of the apical CFTR band observed after apical biotinylation of polarized CFBE41oϪ cell monolayers, and (c) a PKA-dependent activation of CFTR-dependent chloride secretion across the apical membrane. These data could open an important question as to how ⌬F508 CFTR that has escaped from the ER can be redistributed to the apical membrane. This rescue of CFTR-dependent chloride secretion induced by NHERF1 overexpression could be a consequence of various interacting factors: an increase of either ⌬F508 CFTR recruitment to the apical membrane and/or its stability on the apical membrane and an increased recycling efficiency of internalized CFTR, as has been described for ␤2 adrenergic (46,47) and for opioid receptors (48).
It is important to note that, because the total expression levels of ⌬F508 CFTR in CFBE41oϪ cell lysates (the sum of the immature band and the thin mature band) was not significantly different from the total CFTR (mature plus immature bands) in the 16HBE14oϪ cell lysates (75.31 Ϯ 11.02 versus 82.04 Ϯ 10.16 optical density units in CFBE41oϪ and 16HBE14oϪ cells, respectively, n ϭ 7, n.s.), the rescue of the CFTR activity in CFBE41oϪ cells to a level similar to that observed in the non-transfected 16HBE14oϪ cells not overexpressing NHERF1, could be due to a redistribution of ⌬F508 CFTR.
A possible mechanism for this NHERF1-dependent redistribution of ⌬F508 CFTR to the apical membrane may come from the Guggino laboratory (13) where they suggested that NHERF favors surface expression by competing with CAL for CFTR binding. CAL, a CFTR interacting PDZ domain protein that associates mainly with the Golgi apparatus, reduces CFTR expression from the plasma membrane and downregulates mature CFTR enhancing its degradation in the post-Golgi compartments (49). Therefore, these authors suggested that the regulation of CFTR in the plasma membrane involves a dynamic interaction between these two PDZ domain proteins, NHERF and CAL (13). It is possible that in CFBE41oϪ cells, which have a lower NHERF1 expression level than in the normal 16HBE14oϪ cells ( Fig. 2A), some ⌬F508 CFTR that has escaped from the ER is retained within the cytoplasm, while the overexpression of NHERF1 shifts the stoichiometry from CAL to interact with CFTR and thus stimulate the NHERF1⅐CFTR complex to move to the plasma membrane.
In conclusion, our data suggest that the overexpression of NHERF1 may favor the rescue of ⌬F508 CFTR activity in CF affected cells even if the precise molecular and cellular mechanisms remain to be clarified. Several strategies have been shown to overcome the trafficking defects of ⌬F508 CFTR: (a) reduced temperature (50,51); (b) stabilization of the protein by chemical chaperones (52); or (c) disruption of the interaction between ⌬F508 CFTR and molecular chaperones such as calnexin and the heat shock proteins that retain CFTR in the ER (53). The finding that overexpression of NHERF1 can stimulate Cl Ϫ secretion across human airway cells homozygous for the ⌬F508 CF mutation is consistent with the hypothesis that some ⌬F508 CFTR is capable of escaping the degradative pathway and being expressed at the apical membrane in human bronchial epithelial cells. Importantly, because the ⌬F508 CFTR mutant is not a simple trafficking mutant but exhibits multiple defects in stability and activation, the elucidation of the cellular mechanisms that permit NHERF1 to recruit ⌬F508 CFTR to the membrane and rescue its activity could result in more specific therapeutic strategies.