CFBE41o-ΔF (CFBE41o- cells expressing ΔF508 CFTR) cells were routinely maintained in DMEM (Dulbecco’s modified Eagle’s medium) and Ham’s F12 medium (50:50, v/v; Life Technologies) at 37°C in a humidified incubator containing 5% CO₂ as described previously [27]. For low-temperature and delivery of ΔF508-CFTR to the plasma membrane, cells were incubated at 27°C for 24 h or 48 h prior to experiments as indicated. For polarized monolayers, CFBE41o-ΔF cells were seeded onto membrane inserts (Costar, Corning) and cultured on an air-liquid interface for 4 to 5 days before analysis as described previously [10].

Anti-CFTR, mouse monoclonal antibodies (clone 24–1 (ATCC) and MM13-4 (Millipore)) were used as described previously [23]. Mouse anti-AP50/μ2 monoclonal antibody was purchased from BD Transduction Laboratories and used in 1:500 dilutions for immunoblotting. Rabbit polyclonal antibody to mannose-6-phosphate receptor, c-Cbl and Nedd4-2 were purchased from Abcam. Rabbit polyclonal antibody against Dab2 was from Santa Cruz Biotechnology and used in 1:200 dilution. Rabbit polyclonal, anti-CHIP antibody was from Thermo Scientific. Polyclonal anti-β-actin antibody was purchased from Sigma-Aldrich. Alexa Fluor 488-labeled goat anti-mouse IgG antibody and Alexa Fluor 594-labeled goat anti-rabbit IgG antibody were from Invitrogen and used in 1:200 dilution. HRP-conjugated goat anti-mouse IgG antibody and anti-rabbit IgG antibody were from Bio-Rad Laboratories. The SuperSignal West Pico chemiluminescence substrate was purchased from Pierce Chemical Co. The ΔF508 CFTR corrector VX-809 was purchased from Selleck Chemicals. All other chemicals were from Sigma-Aldrich or Thermo Scientific.

siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion^® (Life technologies). Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [11]. The specific sequence 5’-TAGAGCATGAACATCCAGTAA-3’ was chosen as a target for the small interfering RNA (siRNA) depletion of Dab2. The targeting sequence for μ2 knockdown was 5’-TGCCATCGTGTGGAAGATCAA-3’. The targeting sequence for c-Cbl was 5’-CCCGCCGAACUCUCAGAUATT-3’. The targeting sequence for Nedd4-2 knockdown was 5’-CCACAACACAAAGTCACACAG-3’. The double-stranded non-silencing control siRNA sequence 5’-AATTCTCCGAACGTGTCACGT-3’, which has no significant homology to any other genes, was also purchased from Qiagen Inc. Transfection of siRNA oligos was performed using siLentFect lipid reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. Briefly, cells at ~70–80% confluence were transfected with the optimized transfection mixture. After 24 h incubation at 37°C, the transfection mixture was replaced with fresh cell culture medium. Experiments were conducted 3 to 6 days after transfection. The depletion efficiency of individual genes was assessed by Western blotting in each experiment.

CFTR, μ2, Dab2, c-Cbl, CHIP and Nedd4-2 protein levels in control or siRNA-depleted samples were determined as described previously [23]. In brief, about 25 μg cell lysates from each sample were resolved by SDS-PAGE and transferred to PDVF membrane followed by blotting with specific primary antibody and HRP-labeled secondary antibody. The membrane was then developed using chemiluminescent substrate (SuperSignal West Pico, Pierce) and the chemiluminescent signals in the membrane were obtained using a ChemiDoc™ XRS System (BioRad). Densitometry was performed using Image J software.

Indirect immunofluorescence microscopy was performed as described previously [23]. Briefly, cells were seeded onto 12-mm glass coverslips coated with fibrinogen and collagen and cultured at 37°C for 24 h after siRNA transfection. After 24 h of low-temperature (27°C) culturing, cells were incubated at 27°C for 1 h before fixation with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed 3 times for 2 min each with PBS, and then blocked with 2.5% goat serum in PBS. Cells were incubated with primary antibodies diluted in blocking solution for 2 h. Following 3 washing steps with PBS and 0.2% Tween 20, the Alexa Fluor-labeled secondary antibodies (1:200) were applied and incubated 45 min and the coverslips mounted on slides with Vectashield/DAPI (Vector Labs). Microscopy was performed using Leitz epifluorescence microscope. Images were obtained with a cooled, charge-coupled high-resolution camera (Photometrics). IpLab Spectrum software (Signal Analytics) was used for image acquisition. For the ammonium chloride experiments, cells were temperature-rescued for 24 h before they were transferred to 37°C and incubated with growth medium containing 5 mM NH₄Cl for 1 h followed by indirect immunofluorescence microscopy using CFTR and mannose-6-phosphate receptor (M6PR) antibodies.

The expression of cell surface CFTR was measured after biotin-labeling using EZ-link^® Biotin-LC-hydrazide as described previously [28]. Following siRNA transfection and low temperature-rescue, cell surface CFTR was labeled by biotinylation, immunoprecipitation using anti-CFTR antibody (24–1) and then blotted with Avidin D-HRP. rΔF508 CFTR endocytosis was measured in a two-step labeling experiment as described previously [28]. rΔF508 CFTR endocytosis was calculated by the reduction of biotinylated CFTR after a 37°C warm-up period compared to the no warm-up control sample (0 time point) [28].

The cell-surface half-life of rΔF508 CFTR was measured as previously described [29]. Following siRNA transfection and culturing the cells at 37°C for 48 h, CFBE41o-ΔF cells were incubated for an additional 48 h at 27°C to promote ΔF508 CFTR cell surface rescue. During the last 16 h at 27°C, the cells were preincubated with 150 μg/ml of cycloheximide. The cells were then cultured in fresh media containing cycloheximide and incubated at 37°C for 0, 1, 2, 4 and 6 h as indicated. At the end of incubation periods, the cell-surface CFTR was biotinylated using EZ-link^® Biotin-LC-hydrazide as described previously [28]. CFTR was immunoprecipitated from cell extracts using anti-CFTR antibody (24–1) and detected by blotting with avidin D-conjugated horseradish peroxidase and chemiluminescence.

Short-circuit currents (I_SC) were measured under voltage clamp conditions using MC8 voltage clamps and P2300 Ussing chambers (Physiologic Instruments, San Diego, CA) as previously described [23]. CFBE41o-ΔF monolayers were initially bathed on both sides with identical Ringer’s solutions containing (in mM) 115 NaCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.24 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, and 10 D-glucose (pH 7.4). Solutions on both sides were vigorously stirred by bubbling through 95%O₂:5% CO₂ gas. Short-circuit current measurements were obtained using an epithelial voltage clamp. A one-second 3-mV pulse was imposed every 10 s to calculate the resistance by Ohm’s law. Where indicated, the mucosal solution was changed to a low Cl^- solution containing 1.2 mM NaCl and 115 mM Na⁺ gluconate, and all other components as above. Amiloride (100 μM) was added to block residual Na⁺ current, followed by the CFTR agonist forskolin (20 μM) and potentiator genistein (50 μM) as indicated. CFTR_Inh-172 (10 μM) was added to the apical solution at the end of experiments to block CFTR-dependent I_SC. All chambers were maintained at 37°C during experiments.

CFBE41o-ΔF cells were transfected with specific siRNA oligos to deplete adaptor protein or E3 ubiquitin ligases and cultured at 37°C for 48 h. The cells were then switched to 27°C for 48 h to rescue ΔF508 CFTR to the cell surface. During the last 16 h, the cells were treated with 150 μg/ml cycloheximide. The immature form of intracellular ΔF508 CFTR (b-band) was degraded as described previously ([11], data not shown). The cells were then incubated with fresh media containing cycloheximide at 37°C for the time periods indicated to promote endocytosis. To block the endocytosis, 5 μM of Dyngo 4A was added to inhibit the dynamin-mediated internalization. CFTR from the cell lysates were immunoprecipitated using anti-CFTR antibody (24–1) and the ubiquitin level on the CFTR molecules was measured by blotting with a polyclonal anti-ubiquitin antibody (Millipore, Cat. # 09–408, 1:1000 dilution).

All experiments were repeated at least 3 times in duplicate, and the data were expressed as the mean ± standard error. Statistical analysis was performed using student’s t test (2-tailed) in Microsoft Excel (Seattle, WA) and significance was determined at the p<0.05 levels.

To monitor loss of the surface pool of rΔF508 CFTR, we first examined the role of two adaptor complexes on the steady-state levels of rΔF508 CFTR after low temperature rescue. To accomplish this, we performed siRNA knockdown (KD) experiments on the μ2 subunit of AP-2 and Dab2 in human airway epithelial cells expressing ΔF508 CFTR (CFBE41o-ΔF). μ2, one of the four subunits of the AP-2 complex, is known to be required for AP-2 complex formation and function [30]. For the depletion experiments, we determined the maximum depletion conditions for each adaptor. After siRNA knockdown (KD) for 48 h, we cultured the cells for an additional 24 h at 27°C to facilitate ΔF508 CFTR surface expression. The results in Fig 1 illustrate that the μ2 KD efficiency was 90% (Fig 1A) and the Dab2 KD efficiency was 84% (Fig 1B). The amount of maturely glycosylated (C band) rΔF508 CFTR increased ~3 fold in the μ2 KD cells versus ~5 fold in the Dab2 KD cells compared to the control siRNA KD cells (C) (Fig 1A and 1B, respectively). The increase of rΔF508 CFTR after Dab2 KD is consistent with previous studies [26]. Significantly, the level of the core glycosylated ΔF508 CFTR (B band) was also increased proportionately after μ2 or Dab2 KD in comparison to the siRNA control, indicating that the depletion of the adaptors might affect intracellular protein trafficking as shown previously [31, 32]. In addition, using cell surface biotinylation, we compared the surface pools of rΔF508 CFTR following KDs of μ2 and Dab2. We found a ~3-fold increase in cell surface rΔF508 CFTR following the KD of each adaptor compared to control conditions (Fig 1C).

Since the surface rΔF508 CFTR expression levels were affected by the KDs, we next tested if both adaptors were necessary for rΔF508 CFTR endocytosis. For these experiments, we depleted the cells of μ2 or Dab2 and cultured cells as polarized monolayers on filter supports for an additional 4 to 5 days under an air/liquid interface. We monitored rΔF508 CFTR endocytosis at 37°C after it was rescued to the cell surface in low temperature using a surface biotinylation assay (see Materials and Methods section). The level of μ2 or Dab2 depletion was ~90% as measured by immunoblotting (data not shown). The results shown in Fig 2 indicate that both μ2 and Dab2 are required for rΔF508 CFTR endocytosis. The rate of rΔF508 CFTR internalization was reduced in either μ2 or Dab2 depleted cells in comparison to the control during the 7.5 min warm-up at 37°C (Fig 2B). Within 2.5 min warm-up, ~29% of cell surface rΔF508 CFTR was internalized in the control cells. In contrast, only ~4.5% and 8.9% of cell surface rΔF508 CFTR molecules were internalized during the 2.5 min warm-up periods in cells depleted with Dab2 and μ2 respectively (Fig 2C). These results indicate that both μ2 and Dab2 are required for rΔF508 CFTR endocytosis since the KDs result in ~69% and ~83% decreases in endocytosis respectively when compared to the control.

Since rΔF508 CFTR endocytosis was dramatically affected by the KD of μ2 or Dab2, we next tested if either of the adaptors were important for the rapid degradation of rΔF508 CFTR at 37°C by monitoring the cell surface half-life of rΔF508 CFTR. After siRNA KD of each of the complexes and low temperature correction, we switched the cells to 37°C during a cycloheximide blockade and monitored loss of the biotinylatable surface CFTR over time. The results shown in Fig 3 indicate that the surface half-life of rΔF508 CFTR with the control siRNA is 1.1 ± 0.1 h, which is consistent with previous reports (29). The siRNA KD of μ2 had a modest effect on rΔF508 CFTR half-life (1.3 ± 0.2 h), while KD of Dab2 had a more pronounced effect (2.1 ± 0.2 h). The results indicate that μ2 and Dab2 are both necessary for rΔF508 CFTR endocytosis, whereas Dab2 appears to be also important in the post-endocytic down-regulation of rΔF508 CFTR.

At the cell surface wild type CFTR is in a functional complex [33], whereas rΔF508 CFTR is unstable and rapidly degraded [8–10, 12]. Given that Dab2 KD enhanced the surface stability of rΔF508 CFTR, we next tested how the chloride channel activity is affected during the Dab2 KD. In these experiments, we first depleted Dab2 or treated with a control siRNA and 24 h later plated the cells onto filter supports and grew them at an air/liquid interface for 5 days. During the last 24 h the cells were cultured at 27°C to promote ΔF508 CFTR rescue. The monolayers were then mounted in Ussing chambers and analyzed. Forskolin was added to elevate intracellular cAMP levels and activate rΔF508 CFTR. Genistein was added to maximally stimulate the channels and to monitor the non-cAMP activated component of rΔF508 CFTR activity (Fig 4A). The forskolin and genistein stimulated currents were roughly double in the Dab2 depleted monolayers compared to the controls (Fig 4B; 18.8 ± 0.2 compared to 10.5 ± 0.3 μA/cm², respectively). The change in I_sc with the CFTR specific inhibitor, CFTR_inh-172, was also greater in Dab2 depleted monolayers (23.1 ± 1.0 compared to 17.7 ± 1.9 μA/cm², respectively; Fig 4C), although the difference was less pronounced because the Dab2 depleted cells had higher baseline currents, suggesting some degree of pre-activation prior to forskolin and genistein treatment. These results are consistent with elevated surface expression of rΔF508 CFTR during Dab2 depletion, but do not imply that Dab2 depletion increases channel activity through any other mechanism besides simply elevating the surface expression of rΔF508 CFTR.

A number of E3 ligases have been implicated in the down-regulation of wild type or rΔF508 CFTR from the cell surface [11, 17, 34, 35], but whether they affect endocytosis or degradation or both is unclear. Here we separated these two processes and monitored the role of c-Cbl, CHIP, and Nedd4-2 in rΔF508 CFTR endocytosis. Each of the E3 ligases was depleted using siRNA, resulting in more than 90% reduction in their protein levels (Fig 5A). Cell surface levels of rΔF508 CFTR were then tested for each of the KDs and internalization assays were performed on polarized monolayers as described above. The results shown in Fig 5 indicate that the surface pools slightly increased (~2 fold) following the depletion of each of the E3 ligases. The E3 KDs increased the cell surface pools by increasing the amount of protein that may reach the cell surface. All three knockdowns increased CFTR B band and thus allowed for more protein to be rescued by low temperature (data not shown), which is consistent with the results shown by Caohuy et al. in CFPAC-1 cells [34]. More importantly, depletion of each of the E3 ligases had little (CHIP) or no effect (c-Cbl and Nedd4-2) on rΔF508 CFTR endocytosis (Fig 5B and 5C). CHIP depletion did show a modest effect on the endocytosis rate (~17% decrease compared to the control, P = 0.07), but it certainly wasn’t comparable to Dab2 or μ2 depletion. The results suggest that ubiquitination at the cell surface by these E3 ligases do not play a significant role in rΔF508 CFTR endocytosis in polarized human airway epithelial cells.

Next, we tested the effect of depletion of each of the E3 ligases on the surface stability of rΔF508 CFTR. To do this, we first transfected the CFBE41o- ΔF cells with siRNA oligos targeting each E3 ligase followed by low-temperature rescue of ΔF508 CFTR. The cell surface half-life of rΔF508 CFTR was determined at 37°C by biotinylation experiments as descried in the Experimental Methods section after cycloheximide block of protein synthesis. Fig 6 shows that ΔF508 CFTR rescued to the cell surface in the siRNA control sample was rapidly degraded after switching to 37°C, with a half-life ~1.1 h. siRNA depletion of c-Cbl increases the surface half-life to 1.5 ± 0.3 h (Fig 6A), although this change was not significant (P = 0.066). siRNA KD of CHIP had a more pronounced effect and changed the half-life to 2.1 ± 0.6 h (P = 0.02), suggesting that CHIP was required for rΔF508 CFTR down-regulation (Fig 6B). And finally, KD of Nedd4-2 had no significant effect on rΔF508 CFTR since the half-life was not affected (1.1 ± 0.2 versus 1.3 ± 0.1 h; Fig 6C (P = 0.169)).

To determine if combinations of the E3 ligases had additive effects, we measured the cell-surface half-life of rΔF508 CFTR after KD of two or all three E3 ligases. The results shown in Fig 6D demonstrate that the depletion of c-Cbl and/or Nedd4-2 in addition to CHIP KD has no additional effect on the degradation of rΔF508 CFTR. Based on these results, we conclude that the CHIP E3 ligase plays a major role in the surface instability of rΔF508 CFTR in airway epithelial cells. In agreement with the increased half-life of rΔF508 CFTR, the CFTR channel function was also enhanced following CHIP depletion as determined by Ussing chamber experiments (Fig 7A). The forskolin and genistein stimulated currents (ΔI_sc) were increased from 9.97 ± 1.54 μA/cm² in the control to 15.33 ± 2.06 μA/cm² in the CHIP depleted monolayers. Further, the CFTR_inh-172 inhibited ΔI_sc also increased by ~40% in CHIP depleted monolayers (Fig 7B).

Our results above show that both AP2 and Dab2 are involved in the internalization of rΔF508 CFTR from the cell surface, and Dab2 appeared to be important for removal of rΔF508 CFTR from the cell surface. To test this idea, we determined if rΔF508 CFTR delivery to the late endosome/lysosome was inhibited by Dab2 depletion. We depleted Dab2 from cells and cultured the cells at low temperature (27°C) to build up rΔF508 CFTR at the cell surface. Then we compared the distribution of rΔF508 CFTR in control siRNA treated cells and Dab2 depleted cells with a marker of the late endosomal compartment, the mannose-6-phosphate receptor (M6PR). Since lysosomal proteases are delivered to this compartment, proteolytic degradation of proteins begins here [36, 37]. To inhibit the degradation of rΔF508 CFTR in this compartment, we treated the cells with a weak base, ammonium chloride and visualized rΔF508 CFTR in the late endosomal compartment. We have used this technique to monitor transferrin receptor chimeras that were delivered to the lysosomal compartment [37] and wild type CFTR transit through the late endosome [23]. The results shown in Fig 8 demonstrate that under control conditions, rΔF508 CFTR and M6PR do not co-localize (Panel A, merge). However, when cells are treated with the weak base to inhibit proteolysis, co-localization occurs (Panel B, merge, arrowheads), suggesting that rΔF508 CFTR degradation occurs in M6PR-positive compartment. In contrast, Dab2 depleted cells treated with weak base, co-localization is not seen, suggesting that the Dab2 depletion has compromised rΔF508 CFTR delivery to the late endosome, and consistent with the increase in rΔF508 CFTR half-life measurements.

Our data above indicate that two sequential steps are involved in the instability of rΔF508 CFTR: the initial endocytosis of rΔF508 CFTR is mediated through the AP2 and Dab2 complexes and the later post-endocytic targeting of rΔF508 CFTR to the late endosomes and lysosomes is Dab2-dependent. To further delineate the role of Dab2 and the E3 ligases in the cell-surface turnover of rΔF508 CFTR, we measured how the ubiquitination of rΔF508 CFTR is affected. First, we tested how ubiquitination of rΔF508 CFTR takes place in control cells. CFBE41o-ΔF cells were cultured at 27°C for 48 h to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature culturing, 150 μg/ml cycloheximide was added to inhibit protein synthesis and deplete the internal pool of newly synthesized ΔF508 CFTR as described before [9]. The cells were then shifted to 37°C for different time periods to promote endocytosis. As shown in Fig 9A, the ubiquitin levels for rΔF508 CFTR remained constant during the first 7.5 min of 37°C warm-up and then began to increase at the 15 min time point in the control cells. We carefully monitored the temperature of the cells to insure that this lag period was not due to cell temperature effects. The ubiquitin level continued to rise after 15 min until the final time point (60 min). This increase of ubiquitination on rΔF508 CFTR was inhibited in the presence of Dyngo-4a, a dynamin inhibitor, suggesting that the majority of ubiquitin was added to rΔF508 CFTR after internalization. The ubiquitination of rΔF508 CFTR was also largely diminished after CHIP depletion (Fig 9A). Dab2 depletion increases the ubiquitinated pool of rΔF508 CFTR 3-fold. This effect was largely attenuated by CHIP depletion (Fig 9B). Our results suggest that the rΔF508 CFTR localized in the peripheral membrane is first internalized through AP2 and Dab2 and then ubiquitinated by CHIP and targeted to the late endosomes/lysosomes for degradation. Dab2 plays a role in both internalization and lysosomal targeting of rΔF508 CFTR during the peripheral quality control process.

Next, we tested whether the CFTR corrector VX-809 has any effect on stabilizing rΔF508 CFTR on the cell surface. To do this, CFBE41o^- cells expressing ΔF508 CFTR were first cultured for 48 h at 27°C to rescue ΔF508 CFTR to the cell surface. The cells were then transferred to 37°C culture with or without VX-809 and the rΔF508 CFTR molecules that remained on the cell surface were analyzed by biotinylation. As shown in Fig 10A, VX-809 increased the cell-surface half-life (t_1/2) ~2 fold. Furthermore, the polyubiquitination of rΔF508 CFTR was reduced by VX-809 treatment (Fig 10B). These results demonstrated that the corrector VX-809 stabilizes the rΔF508 CFTR at the plasma membrane in addition to its function of promoting ΔF508 CFTR folding in the ER compartment.