Abstract
Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed ∼450 unique proteins fused to the Cl−-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI−/l− exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3α, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens.
Cystic fibrosis (CF)1 is the most common genetic disorder in the Caucasian population, affecting 1:2500 live births. It is caused by mutations in the CFTR gene, which encodes a cAMP-regulated Cl− channel (reviewed in (1, 2). Although several classes of mutation in CFTR have been identified to date (1), the most common mutation found in patients of European ancestry is a deletion of phenylalanine at position 508 (F508del-CFTR) (3). The F508del-CFTR mutant is a trafficking impaired mutant that is retained in the endoplasmic reticulum (4–6), thus its absence from the plasma membrane precludes Cl− secretion, leading to CF. Partial correction of this trafficking defect can be obtained by lowering the temperature (e.g. 27 °C) or treating cells with glycerol (7, 8). These maneuvers, however, cannot be used to treat patients. Thus, over the past few years, several groups have developed high-throughput screens to identify small molecules that can correct the trafficking and functional defects of the F508del-CFTR mutant, such as compounds 3a and 4a (9–13), carboplatin, sildenafil or its analogues (14, 15), VRT-325, and VRT-640 (16, 17). Some of these compounds (e.g. VRT(VX)-809 or VX-770) are now in pre-clinical trials.
While identifying small molecules that correct the trafficking defect of F508del-CFTR can be very valuable as a clinical tool; such an approach does not identify the cellular protein or pathway that is targeted by the small molecule/compound. Thus, we embarked on the development of a high-content/high-throughput functional assay that allows for the identification of proteins that correct F508del-CFTR function in multiple individual cells simultaneously, using Cellomics KineticScan technology. We generated a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR and individually co-expresses several hundred proteins fused to the Cl−-sensitive YFP mutant, YFP(H148Q/I152L) (12, 18), to test for Cl− transport via CFTR in a high-content/high-throughput manner. Here we describe the development of the assay and the identification of proteins that when co-expressed with F508del-CFTR help rescue its function by enhancing its maturation.
EXPERIMENTAL PROCEDURES
Media and Reagents
Dulbecco's modified Eagle's medium, F12 nutrient mixture, Opti-MEM I reduced-serum medium, Dulbecco's phosphate-buffered saline (D-PBS) with and without calcium or magnesium, fetal bovine serum (FBS), trypsin, and Lipofectamine 2000 were obtained from Invitrogen. Methotrexate was from Sigma, and fluorescent mounting medium was from DakoCytomation. Propidium iodide and rhodamine-conjugated concanavalin A were purchased from Invitrogen. Normal goat serum was from Cederlane. SuperSignal West Femto Maximum Sensitivity kit was from Pierce. The corrector compound 4a (corr-4a) was obtained from the Cystic Fibrosis Foundation Therapeutics (CFFT) library (kindly provided by Dr. R. Bridges) and Velcade was from Millenium Pharmaceuticals. Mouse anti-HA.11 monoclonal antibody (MMS-101R) and anti-GFP antibody (MMS-118R) were from Covance, and Alexa Fluor 647-labeled goat anti-mouse antibody was from Invitrogen (A21236). The mouse M3A7 anti-CFTR monoclonal antibody was obtained from Chemicon (MAB3480) and the anti-β-actin monoclonal antibody was from Sigma (A5441). The rabbit polyclonal anti-PIAS1 antibody (ab58403) and the mouse monoclonal anti-AHA1 antibody (H00010598-M01) were from Abcam and Abnova, respectively.
Expression Vector Constructs and esiRNA
A new destination vector was generated by replacing the V5 epitope with eYFP(H148Q/I152L) in the pre-existing Gateway destination vector pcDNA3.1/nV5-DEST (Invitrogen). 446 clones (all from the Gateway ORFeome v1.3) were then cloned by recombination into this new destination vector (called PCDNA3.1(eYFP H148Q/I152L)). For assay validation DNA sequences of 9 randomly picked hits and 3 negative control proteins were shuttled into pcDNA6.2/N-emGFP destination vector using Gateway technology. esiRNA for PIAS1 and AHA1 knockdowns was prepared as described previously (19).
Cells
HEK293 MSR GripTite (293MSR-GT) cells (Invitrogen) were stably transfected with C-term-VSVG-tagged wild type or F508del-CFTR cDNA in pLenti6 vector using calcium phosphate method. At 24-h post-transfection, the cells were split and selected under 25 μg/ml blasticidin. Individual clones were picked and expanded. Expression of wild type CFTR or its mutant F508del-CFTR was validated by immunoblotting using M3A7 anti-CFTR or anti-VSVG monoclonal antibodies. 293MSR-GT cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1× non-essential amino acids, 0.6 mg/ml G418 and 10 μg/ml blasticidin at 37 °C, 5% CO2 in humidified atmosphere. Baby hamster kidney (BHK) cells stably expressing wild type (CFTR-3HA) or mutant (F508del-CFTR-3HA) protein with the triple hemagglutinin (3HA) tag at the ectodomain were a kind gift from D. Y. Thomas (McGill University, Montreal). Cells were propagated as monolayer cultures in Dulbecco's modified Eagle's medium-F12 medium 1:1 supplemented with 5% FBS and 0.5 mm methotrexate at 37 °C, 5% CO2. The cells were maintained in the growth medium for a maximum of 8 weeks or 16 passages.
Transient Cell Transfection
For the screen, 60,000 293MSR-GT cells (stably expressing F508del-CFTR) per well were seeded in the 96-well plates (in duplicates). The next day the cells were transfected on both plates with 400 ng of DNA using the calcium phosphate method and incubated at 37 °C. At 24-h post-transfection one plate was transferred to 27 °C, and the other was kept at 37 °C for additional 24 h. For the esiRNA assay, 40,000 293MSR-GT cells (stably expressing F508del-CFTR and YFP H148Q/I152L) were seeded and transfected the next day with esiRNA for PIAS1 and Aha1 using Lipofectamine 2000. For live cell staining, BHK F508del-CFTR-3HA cells were plated in p100 dishes to achieve 80% confluency the next morning. The cells were then transfected using Lipofectamine 2000 with 6 μg of DNA. For Western blotting analysis or staining of the fixed non-permeabilized cells, cells were grown in p100 plates or on microscope glass coverslips in 6-well plates to achieve 80% or 60% confluency, respectively. On the day of transfection cells were transfected using Lipofectamine 2000 according to manufacturer's instructions.
Cellomics High-content Assay/Screen
A total of 446 proteins were tested, and each test protein was run in triplicate wells in 96-well plates, in a temperature-controlled chamber with 5% CO2. At 48-h post-transfection the medium was replaced with 152 μl of chloride solution (137 mm NaCl, 2.7 mm KCl, 0.7 mm CaCl2, 1.1 mm MgCl2, 1.5 mm KH2PO4, 8.1 mm Na2HPO4, pH 7.1), in the absence or presence of forskolin/IBMX/genisein (FIG) (25 μm forskolin, 10 μg/ml IBMX, 50 μm genistein), or the inhibitors 172 (100 μm) or glybenclamide (2 μg/ml), at 37 °C or 27 °C, as indicated. After 20 min of incubation, 92 μl of I− iodide buffer (137 mm NaI, 2.7 mm KCl, 0.7 mm CaCl2, 1.1 mm MgCl2, 1.5 mm KH2PO4, 8.1 mm Na2HPO4, pH 7.1) was added (final-concentration of 52 mm), and decrease in fluorescence over time was recorded using the Cellomics KineticScan (ThermoFisher), at 27 °C. The same assay was also performed on cells rescued at 27 °C (24 h) or treated with 1 or 10 μm compound 4a (corr-4a) (18 h), or 1 or 10 μg/ml Velcade (18 h). Images for 100–300 transfected cells/well were acquired simultaneously using the XF93 filter set (Omega Optical, Brattleboro, VT) X20 objective (0.4 NA). The microscope was set to autofocus.
Image Analysis
The Cellomics software uses an object-identification algorithm based on intensity thresholds between adjacent pixels. Identified objects within an imaged field were accepted or rejected for analysis on the basis of fluorescent intensity. Total fluorescence intensity within the accepted region was determined for eYFP(H148Q/I152L) over a 36-s time course. A modified Target Activation BioApplication (version 2; Cellomics) software was used for data analysis of eYFP(H148Q/I152L) quenching. Data were exported to Microsoft Excel. Proteins that exhibited rescue of at least 0.25 (25%) (i.e. difference in average normalized fluorescence between FIG alone and FIG + test protein ≥ 0.25) were considered “top” hits, and those exhibiting rescue between 20–25% were considered “second tier” hits. For each hit, the experiment was repeated 2–3 times (each in triplicates).
Validation of Rescue of the F508del-CFTR Mutant
The cDNA for all top hits and controls were sequence-verified to re-confirm identity.
Immunoblotting—
At 48-h post-transfection the cells were rinsed in PBS and lysed in lysis buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol (v/v), 1% Triton X-100 (v/v), 2 mm phenylmethylsulfonyl fluoride, 2× PAL inhibitors). Proteins were resolved on SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-CFTR monoclonal antibodies (M3A7, 1 μg/ml), anti-β-actin antibodies (1:10000), anti-AHA1 antibodies (1:1000), anti-GFP antibodies (1:2000), or polyclonal anti-PIAS1 antibodies (1:1000). Membranes were washed with 5% Blotto, incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:5000 in 3% skim milk) and washed with PBS-Tween. Signal was detected with SuperSignal West Femto reagent.
Confocal Microscopy—
60 h after transfection, cells were washed with PBS, fixed with 10% phosphate-buffered formalin, and washed again. They were then stained with ConA-Rhodamine (1:500 dilution, 5 min), washed, and incubated with 5% normal goat serum and 1.5% bovine serum albumin for 30 min. The anti-HA.11 monoclonal antibody was added (4 μg/ml in the blocking buffer) and incubated at room temperature for 1 h. Cells were washed with PBS and stained with AF647-labeled goat anti-mouse antibody (2 μg/ml) for 1 h. The coverslips were washed and mounted on microscope slides. Stained cells were then analyzed by Zeiss LSM510 laser scanning confocal microscope.
Flow Cytometry—
At 48-h post-transfection cells were trypsinized, washed, and re-suspended in ice-cold FACS buffer (PBS supplemented with 2% FBS). To stain the cell surface, cells were incubated with anti-HA.11 monoclonal antibody (20 μg/ml) or AF647-labeled goat anti-mouse antibody (10 μg/ml) as a control, for 1 h at 4 °C. Subsequently the cells were washed with the cold FACS buffer and incubated with AF647-conjugated goat anti-mouse antibody (10 μg/ml) at 4 °C for 1 h. They were then washed as above and re-suspended in FACS buffer with 1 μg/ml propidium iodide. The flow-cytometric analysis was performed using FACSCalibur System (BD Biosciences). The data from 10,000 GFP positive cells were stored and analyzed with FlowJo software.
RESULTS
Development of the High-content/High-throughput Assay for Analysis of Function of CFTR or Its Mutants—
To develop the high-content functional screen for identification of correctors of F508del-CFTR, we generated cell lines in which wild type (WT) CFTR or F508del-CFTR (both tagged with VSVG at the C terminus) were stably transfected into 293MSR-GT cells, which were human cells. The use of 293MSR-GT cells was necessary to increase adherence of the cells in order to perform washes and addition of solutions carried out robotically. Expression of WT and F508del-CFTR in these cells was verified by immunoblotting with antibodies to CFTR (Fig. 1A) or VSVG (not shown). In parallel, we generated the Cl− sensitive YFP mutant, YFP(H148Q/I152L) (12, 18), in the Gateway protein expression destination vector (Invitrogen) and verified its expression in the 293MSR-GT cells (Fig. 1B). We then shuttled hundreds of cDNA clones into this vector from the human ORFeome library (see below).
Generation of WT and F508del-CFTR stably expressed in 293MSR-GT cells.A, immunoblot showing WT and F508del-CFTR stably expressed in 293MSR-GT cells, which was detected with anti-CFTR antibodies. Note the presence of band C (mature CFTR) in the WT-CFTR as well as in temperature-rescued (27 °C) F508del-CFTR. B, fluorescence quenching reflecting CI−/l− exchange of YFP(H148Q/I152L) in 293MSR-GT cells expressing WT (top panel), F508del-CFTR (middle panel), or temperature-rescued F508del-CFTR (lower panel). Cells were treated for 20 min with FIG and then exposed to high I−/low Cl− medium for 36 s to follow CI−/l− exchange (CFTR activity), reflected by fluorescence quenching. Fluorescence was recorded on the Cellomics high-content microscope.
To develop the assay, 293MSR-GT cells stably expressing F508del-CFTR (F508del-CFTR cells) or WT-CFTR (CFTR cells) were transfected with the YFP(H148Q/I152L) construct. Two days after transfection, cells were stimulated for 20 min with forskolin (25 μm)/IBMX (10 μg/ml)/genistein (50 μm) mixture (FIG). They were then exposed to low Cl−/high I− medium by adding iodide solution to a final I− concentration of 52 mm (and maintaining normal osmolality), and fluorescence quenching of transfected cells due to Cl−/I− exchange (presumably via CFTR) was monitored and quantified over time by the Cellomics KineticScan reader, as depicted in Fig. 1B (top panel) and Fig. 2A. To test the ability of the assay to detect rescue of F508del-CFTR, the same assay was performed on F508del-CFTR cells that were pre-incubated at 27 °C overnight prior to the assay (temperature rescue). As seen in Fig. 1B (bottom) and Fig. 2B, the assay detected a robust rescue of F508del-CFTR at low temperature, which was not detected at 37 °C (Fig. 1B, middle panel). As expected, quenching of fluorescence (i.e. Cl−/I− exchange) was inhibited with known inhibitors of CFTR, such as glybenclamide or compound 172 (10, 20) (Fig. 2, A and B). Moreover, our assay was able to detect the rescue of F508del-CFTR by a recently described corrector, compound 4a ([2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]bithiazolyl-2′-yl]-phenyl-methanone) (13) at 1 μm (not shown) or 10 μm (Fig. 2C). The small potentiation of F508del-CFTR function in response to FIG treatment alone (e.g. Fig. 2C) likely reflects the residual Cl− channel activity of the overexpressed (stably transfected) F508del-CFTR.
Quantitative analysis of the high-content (Cellomics) assay. Average normalized fluorescent values of WT-CFTR or F508del-CFTR expressed in 293MSR-GT cells (which co-expressed YFP(H148Q/I152L) that were grown at 37 °C or 27 °C, as indicated. Fluorescent quenching during CI−/I− exchange of 100–300 cells is quantified simultaneously and recorded. A, WT-CFTR grown at 37 °C were unstimulated or stimulated with FIG alone, FIG + the CFTR inhibitor 172 (100 μm), or FIG + the CFTR inhibitor glybenclamide (Glyb, 2 μg/ml). B, rescued (27 °C) F508del-CFTR unstimulated or stimulated with FIG or FIG + 172. C, F508del-CFTR grown at 37 °C and stimulated with FIG + corrector 4a (10 μm), a known corrector of F508del-CFTR. Data are average of triplicate wells, with 100–300 cells analyzed per well.
Together, these results suggest that the high-content quantitative assay we developed is sensitive enough to detect correction of F508del-CFTR function and hence is amenable to performance of high-content/high-throughput screen to identify proteins that correct the function of F508del-CFTR.
Identification of Proteins that Correct F508del-CFTR Function Using the High-content Assay—
Once we established the high-content assay for F508del-CFTR rescue, we proceeded to screen 446 proteins as a pilot study to test whether any of them could rescue F508del-CFTR function when co-expressed with the F508del-CFTR (at 37 °C). The cDNAs encoding the proteins chosen for this pilot study (listed in supplemental Table S1) included chaperones, trafficking proteins, proteins known or previously proposed to interact with CFTR (CFTR interactors), proteins involved in the ubiquitin system, signaling proteins, as well as randomly chosen proteins. The cDNAs for the proteins, all from the Gateway human ORFeome library (21) were shuttled into the above described YFP(H148Q/I152L) mammalian expression vector (Gateway compatible destination vector), such that each protein was expressed as a fusion-protein with the YFP(H148Q/I152L) moiety. Each of these cDNAs was then transfected into the F508del-CFTR cells in a 96-well format (in triplicates) and FIG-induced CI−/I− exchange tested 2 days later using the Cellomics high-content assay described above, at 37 °C (5% CO2). Fig. 3 (B–D) depicts examples of several “hit” proteins that when co-expressed with F508del-CFTR exhibited correction of its function, whereas Fig. 3A provides an example of a protein (CLIC4) that did not correct F508del-CFTR function, shown here for comparison. The list of all the “top hits” (defined as those exhibiting at least 25% rescue) is provided in Table I, and the extent of their correction is quantified in Fig. 3E. This hits list reveals several correctors that are known to be involved in protein maturation, processing, and trafficking, several signal transduction proteins and other proteins. A list of proteins that exhibited a lower extent rescue (“second tier”, defined as those exhibiting 20–25% rescue) is provided in supplemental Table S2. Both the top hits as well as the “second tier hits” revealed greater extent of rescue than compound 4a in our assay.
Top hits of the high-content screen. 293MSR-GT cells stably expressing F508del-CFTR were either transfected with YFP(H148Q/I152L) alone (control) or with the indicated cDNAs (test protein) fused to YFP(H148Q/I152L) and grown at 37 °C. They were then treated with FIG for 20 min and subsequently subjected to CI−/I− exchange for 36 s. A, example of a non-corrector (CLIC4). B–D, examples of three of the top correctors, as indicated. E, quantitation of rescue of all the top hits at 36 s after adding I− solution (normalized average fluorescence (Cl−/I− exchange) from 2–3 experiments, each performed in triplicates). 4a, corrector 4a (10 μm). Data are mean ± S. E.
List of the top hits identified in the screen
Top hits were defined as proteins with difference in average normalized fluorescence of ≥0.25 between the FIG treatment alone and FIG + test protein (see Fig. 3).
Validation of the Top Hits by Independent Methods—
To further analyze and validate the hits, we employed alternative methods to demonstrate rescue of F508del-CFTR by our top hit proteins, randomly choosing 9 of them. For these experiments, we utilized both our F508del-CFTR expressing 293MSR-GT cells, as well as BHK cells that stably expressed F508del-CFTR, which contained a triple HA tag at its ectodomain (kindly provided by D. Thomas, McGill University, Montreal) (14). First, we tested for the appearance of a mature F508del-CFTR protein represented by band C in a Western blot. Our results show that co-expression of these top hit proteins with F508del-CFTR in 293MSR-GT cells (Fig. 4) or BHK cells (not shown) led to the appearance of the mature band C, similar to that observed following low temperature (27 °C) treatment or exposure to compound 4a. Band C was not detected in controls A1, B1, and C1 (ZCCHC9, MGC4677, and DPM2, respectively), which were proteins that did not rescue F508del-CFTR in our screen.
Validation of the hits by immunoblotting for mature F508del-CFTR in 293MSR-GT cells. 293MSR-GT cells stably expressing F508del-CFTR were transfected with the cDNAs corresponding to some of the top hit proteins (each GFP-tagged), as indicated, grown at 37 °C, and the appearance of the mature protein, band C, monitored by immunoblotting with anti-CFTR antibodies. Band B represents the immature protein. The transfection efficiency of the GFP-tagged hits was determined by fluorescent microscopy and shown to be at least 85%. Controls 1–3 are proteins that did not rescue F508del-CFTR in our assay (ZCCHC9 (A1), MGC4677 (B1), DPM2 (C1)), respectively (see supplemental Table S1); GFP represents GFP alone, and 27 °C represents temperature rescue of F508del-CFTR at 27 °C. The figure depicts the anti-CFTR immunoblot (top), as well as actin (loading) control (middle), and anti-GFP controls (bottom) to show the amount of transfected (GFP-tagged) proteins expressed in the cells. The expression of GFP alone and Control A1 is not seen in this blot due to its small size.
Next, we tested the appearance of F508del-CFTR at the plasma membrane of non-permeabilized BHK cells. The cells were transfected with the hit proteins cDNAs and immunostained for the exofacial HA tag followed by confocal microscopy analysis. For this experiment, the hit proteins were expressed fused to GFP. Fig. 5 (C and D) shows two examples of such hits, endothelin 1 (ET-1) and STAT1, revealing that their co-expression with F508del-CFTR at 37 °C led to the appearance of F508del-CFTR at the cell surface, similar to the rescue of F508del-CFTR by glycerol (not shown) or low temperature (Fig. 5B). As expected, F508del-CFTR alone (at 37 °C) was not detected at the cell surface (Fig. 5A).
Validation of hits by immunofluorescence confocal microscopy. BHK cells stably expressing F508del-CFTR-3HA (bearing triple HA tag at the ectodomain) were either untranfected (A and B) or transfected with GFP alone (E) or with the GFP-tagged ET-1 (C) or STAT1 (D). Non-permeabilized cells were then immunostained with anti-HA antibodies to detect cell surface expression of F508del-CFTR (white), as well as Rhodamine-conA (red) to mark the plasma membrane. Transfected cells expressing GFP are green. B, demonstrates temperature (27 °C) rescue of F508del-CFTR, which is not seen at 37 °C (panel A).
Since the appearance of F508del-CFTR at the plasma membrane (stimulated by the co-expressed hit proteins) that was analyzed by confocal microscopy could not be accurately quantified, we used flow cytometry to quantify and compare the amounts of F508del-CFTR at the cell surface following co-expression of the 9 hit proteins. Thus, GFP-tagged proteins were expressed in BHK cells that stably expressed the F508del-CFTR-3HA, and the amount of cell surface F508del-CFTR (i.e. HA) was quantified 2 days later (at 37 °C). Fig. 6A depicts low temperature (27 °C) rescue of F508del-CFTR analyzed by this flow cytometry approach, whereas Fig. 6B demonstrates lack of such rescue with a non-rescuing control protein (DPM2) at 37 °C. As seen in Fig. 6, C–E and summarized in G, different degrees of rescue were obtained by the co-expressed hit proteins. Interestingly, co-expression of STAT1 strongly stimulated the appearance of F508del-CFTR at the cell surface, with better effect than compound 4a (Fig. 6, C and F). In support, we found that knockdown of PIAS1, a known inhibitor of STAT1, also promoted rescue of F508del-CFTR (supplemental Fig. S1). ET-1 also provided good levels of F508del-CFTR rescue. Collectively, these results suggest that the high-content functional and quantitative assay we developed for identification of proteins that rescue F508del-CFTR is a valid and robust assay.
Validation of hits using flow cytometry. BHK cells stably expressing F508del-CFTR-3HA were transfected (or not) with the indicated hit cDNA fused to GFP. Flow cytometry was then performed on non-permeabilized cells following immunostaining for the HA epitope located at the ectodomain of F508del-CFTR, to quantify the amount of cell-surface F508del-CFTR in the transfected (GFP-expressing) cells. A, temperature (27 °C) rescue of F508del-CFTR (blue line). Red line, 37 °C. B, control (DPM2, a non-rescuing protein from the screen). C–E, hit proteins STAT1, ET-1, and LGALS3. F, rescue with corrector 4a (blue line). Red line, 37 °C control. C–E, red line, GFP alone. Blue line, GFP-tagged corrector protein. G, summary of increase in cell surface expression of F508del-CFTR (% change in fluorescence intensity) of all the top hits (mean ± S. E., n = 2–3 independent experiments).
Rescue of 508del-CFTR with Velcade—
One advantage of our approach of identifying proteins that rescue F508del-CFTR is that if a known stimulator drug of any hit protein already exists in the clinic for other uses, it can be now tested and possibly applied for rescuing F508del-CFTR. To this end, the proteasome inhibitor Velcade (Bortezamib), used clinically to treat multiple myeloma, has been recently demonstrated to enhance the cellular stress response, including Hsp70 expression (22, 23). Hsp70 is a chaperone related to HspA4, and is shown to enhance F508del-CFTR maturation (24). Since HspA4 was identified in our screen as one of the top hits, and several other chaperones were also identified as rescuers (e.g. HspBP1, DnaJ, HSPB8, CRYAB, Park7/DJ-1; supplemental Table S2), we tested whether Velcade can rescue F508del-CFTR. As seen in Fig. 7A, incubating the 293MSR-GT cells that stably expressed F508del-CFTR led to the rescue of this mutant similar to that seen with overexpressed HspA4 or other hit proteins. This rescue was also validated by the appearance of band C (mature F508del-CFTR) in a Western blot (Fig. 7B, bottom panel). In addition, a clear effect of the Hsp90 chaperone system on maturation of F508del-CFTR was recently demonstrated (25). In agreement, our Cellomics halide exchange assay demonstrated rescue of F508del-CFTR function by reduction of Aha1 levels with esiRNA (supplemental Fig. S1).
Rescue of F508del-CFTR with Velcade.A, F508del-CFTR stably expressed in 293MSR-GT cells were treated (or not) with 1 or 10 μg/ml Velcade overnight prior to performing the Cellomics assay described in the legend to Fig. 2, with or without stimulation with FIG. Inset (box), normalized rescue of F508del-CFTR to FIG-treated cells. Data are mean ± S. D. (n = 6), or data are mean ± S. E. (n = 6, error bars are smaller than the data points). B, immunoblotting for mature F508del-CFTR (band C) using anti-CFTR antibodies in 293MSR-GT cells stably expressing F508del-CFTR after Velcade treatment, as described in the legend to Fig. 4.
Use of the High-content Assay to Identify Inhibitors of F508del-CFTR Rescue—
The high-content assay we developed can also detect inhibition of CFTR function, which can provide useful information for the treatment of diarrhea. It can also identify proteins that interfere with F508del-CFTR rescue. That information can be useful for future targeting of these inhibitors, for example with small molecules. In our assays described above, the same 446 proteins (supplemental Table S1) were also tested if they could inhibit of F508del-CFTR rescue at 27 °C. Fig. 8 depicts two examples of such inhibitors, the de-ubiquitination enzymes USP47 and UCHL5.
Inhibition of temperature rescue of F508del-CFTR. F508del-CFTR stably expressed in 293MSR-GT cells were transfected with cDNAs fused to YFP(H148Q/I152L), as listed in supplemental Table S1. They were subsequently incubated overnight at 27 °C and then tested for inhibition of temperature rescue of F508del-CFTR using the Cellomics assay as described in the legend to Fig. 2. The extent of inhibition of the hit protein (A) USP47 or (B) UCHL5 was compared with a known inhibitor of CFTR, compound 172. Data are average of 3–5 wells (each with 100–300 cells analyzed) per condition.
DISCUSSION
In this paper, we describe the development of a high-content/high-throughput functional screen to identify proteins that when ectopically expressed with F508del-CFTR (at 37 °C) correct its function. This assay can also be used to identify proteins that inhibit CFTR function (or inhibit rescue of F508del-CFTR at 27 °C), for RNAi screens, small molecules, or peptide screens. Given the ability of the Cellomics to simultaneously acquire data from cells that express the transfected protein (e.g. cells expressing YFP or GFP), but not surrounding untransfected cells, the use of this type of assay is particularly suitable for screens (e.g. RNAi screens) that utilize hard to transfect cells, such as primary cells. In addition, the advantage of our screen, which focuses on proteins rather than on small molecules, is that it can identify novel pathways in which CFTR participates.
Some of the proteins that we tested were previously shown to affect CFTR maturation (e.g. HspBP1, HspA4 (related to Hsp70) or the Hsp90 co-chaperone Aha1) (25–27). Several of these were also identified as promoting F508del-CFTR rescue in our screen. However, some other proteins were missed by our screen. There are several possible explanations for this: (i) their levels of transfection/expression were not high enough; (ii) their cellular levels are already maximal such that further increase in their amounts would not have an effect; or (iii) their effect is below our pre-determined cutoff point.
The correctors we identified include several chaperones (e.g. HspA4, HspBP1, CRYAB, HspB8, DnaJ and Park7/DJ-1), Golgi-associated proteins and trafficking proteins (e.g. Cav2, GORASP, LGALS3, AP2M1, GAPDH, PKCi), signaling proteins and transcription factors (e.g. STAT1, ET-1, PKCi, DUSP3, SAKS1, TFG, TCP10L), proteins involved in the ubiquitin system, and proteins with unknown function (see Table I and supplemental Table S2). Significantly, in our assays, all of these proteins yielded better correction than the previously described corr-4a, a well known corrector of F508del-CFTR (13). One of the best F508del-CFTR rescuing protein we identified is STAT1 (Signal Transducer and Activator of Transcription 1), which is a key component in the signal transduction downstream of cytokine receptors (28). In support, knockdown of PIAS1 (a known inhibitor of STAT1) with esiRNA also rescued F508del-CFTR. Earlier work had demonstrated that in colonic epithelial cell lines IFNγ-mediated signaling via STAT1 leads to a reduction in CFTR expression (29, 30). Intriguingly, in CF epithelial cells, despite aggressive inflammation, expression of PIAS1 is elevated, leading to a reduction in STAT1 and NOS2 (the enzyme responsible for nitric oxide production) activation; this process likely involves activation of RhoA and Rock upstream of PIAS1 (31, 32). The possible role of STAT1 protein in rescuing F508del-CFTR has not been recognized until now and thus brings a new dimension to our understanding of the interplay between activation of the immune system so prevalent in CF and the efforts made by the affected cells to correct the underlying defect.
In addition to STAT1, among the signal transduction proteins identified as strong correctors of the F508del-CFTR defect was ET-1. ET-1 has been demonstrated to rapidly increase CFTR activity in ventricular myocytes (33), and our work here shows that it also stimulates trafficking of F508del-CFTR to the plasma membrane. Interestingly, ET-1 production is elevated in CF lungs (34), suggesting a compensatory mechanism to promote an increase in cell-surface amounts and activity of the mutated CFTR. This putative compensation, however, may come at a price, as ET-1 is also a powerful bronchoconstrictor.
Besides ET-1, among the identified protein correctors not previously known to affect CFTR maturation were pancreatitis-associated protein 1 (PAP/REG3α) and calgranulin A (S100A8). PAP is a C-lectin stress response protein with an anti-apoptotic activity the expression of which is elevated in the pancreas of CF mice (35) and in CF patients (36, 37). The identification of S100A8 (a “CF antigen”) was interesting as well because it is known to be up-regulated in response to inflammation in the lung that is usually associated with CF (38–40).
It is worth noting that our screen also identified proteins that when overexpressed inhibited maturation of F508del-CFTR at 27 °C. The two proteins that we identified (USP47, UCHL5) are both de-ubiquitination enzymes. The function of USP47 is so far unknown, but UCHL5 (USP37) associates with the proteasome and is responsible for removal of polyubiquitin chains from condemned proteins prior to their entry into the 20 S proteasome for degradation (41, 42). It is possible that overexpression of UCHL5/USP37 (and possibly USP47) may lead to enhanced degradation of F508del-CFTR, which is poly-ubiquitinated due to its misfolding, resulting in reduced rescue of this mutant protein.
Although some of the proteins identified in the screen were previously known or suspected to interact with and/or affect CFTR function, as described above, the rest of the hits are novel (see supplemental Table S3), and the mechanisms by which they may modulate F508del-CFTR trafficking and function are not known and await future investigations.
Identifying small molecules that correct the maturation defect of F508del-CFTR may be very valuable as a clinical tool. Nevertheless, such an approach does not identify the cellular protein or pathway that is targeted by the small molecule/compound. Our screen can identify novel pathways in which CFTR or F508del-CFTR participate, and thus if clinically approved drugs (used to treat other diseases) are known to modulate components of the pathway, they can then be tested for correction of F508del-CFTR function. Indeed, our (and other groups) identification of HspA4, HspBP1, and other chaperones as proteins when overexpressed correct F508del-CFTR cell surface expression and function led us to test the ability of Velcade (a proteasome inhibitor that activates the cellular stress response, including stimulation of chaperones) to rescue F508del-CFTR. Our work here shows that Velcade can indeed correct F508del-CFTR function, in accordance with a recent report by Zeitlin and colleagues (43) and with a report that demonstrates rescue of mutant CFTR with the proteasome inhibitor LLnL (44). Since Velcade is already used clinically for the treatment of multiple myeloma, it may be possible to test its efficacy for treating CF. It is interesting to note that Velcade stimulated F508del-CFTR function even in the absence of FIG treatment, suggesting that it may function both as a corrector and potentiator of F508del-CFTR. Similarly, given our identification of STAT1 as a corrector of F508del-CFTR cell surface expression and function, it would be worthwhile testing the effect of interferons, known to activate STAT1, on rescue of F508del-CFTR. Although STAT1 can be involved in several signal transduction pathways (28), our observation of rescue of F508del-CFTR upon knockdown of PIAS1 and the previously documented elevation of PIAS1 levels in CF epithelium via the RhoA/Rock (Rho kinase) pathway (32), raises the possibility that the RhoA/Rock pathway is involved in the STAT1-mediated rescue of F508del-CFTR. Thus, it would be worthwhile testing the effect of statins (cholesterol-lowering drugs (HMG-CoA reductase inhibitors), which inhibit RhoA) on rescue of F508del-CFTR. In support, Kelley and colleagues reported that Mevastatin corrects the reduced STAT1 signaling in CF cells by inhibiting PIAS1 (32).
The various correctors (hits) we identified corrected F508del-CFTR to different degrees, which could reflect their levels of overexpression or effectiveness at rescue. Since it has been proposed that correction of even ∼10% of CFTR activity may be sufficient to restore airway epithelial function (45), the identification of even partial rescue of F508del-CFTR is significant. Moreover, since CF patients exhibiting residual Cl− channel activity have milder disease (46, 47), such partial rescue of F508del-CFTR function may have therapeutic benefits.
In summary, our work here has demonstrated the development of a high-content assay to identify correctors of F508del-CFTR and the identification of several known and novel proteins that when co-expressed with F508del-CFTR lead to rescue of its trafficking and functional defects. Future work will delineate the mechanisms by which these novel modulators bring about rescue of F508del-CFTR, as well as the effect of these proteins on primary cells from CF patients.
Acknowledgments
We thank Dr. Bart Kus and Cheryl Wolting for the initial work on assay development and data analysis (Python program), Kevin Yoong for database searches, Dr. D. Y. Thomas (McGill) for the CFTR-expressing BHK cells, and Drs. C. Bear, F. Ratjen, and P. Durie for critically reading the manuscript.
Footnotes
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Published, MCP Papers in Press, December 15, 2008, DOI 10.1074/mcp.M800268-MCP200
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↵ 1 The abbreviations used are: CF, cystic fibrosis; 293MSR-GT, HEK293 macrophage scavenger receptor GripTite cell; FIG, forskolin/IBMX/genistein; CFTR, cystic fibrosis transmembrane conductance regulator; RNAi, RNA interference; esiRNA, endoribonuclease-prepared siRNA; ConA, concanavalin A; PBS, phosphate-buffered saline; FBS, fetal bovine serum; BHK, Baby hamster kidney; ET-1, endothelin 1; HA, hemagglutinin; WT, wild type; PAP, pancreatitis-associated protein 1; GFP, green fluorescent protein; YFP, yellow fluorescent protein; VSVG, VSVG tag.
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↵* This work was supported by the Canadian Cystic Fibrosis Foundation/Canadian Institutes of Health Research (CIHR) (BREATHE program) and the Canadian Foundation for Innovation/Signaling Identification Network (SIDNET).
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↵S The on-line version of this article (available at http://www.mcp.org) contains supplemental material.
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↵‡ Both authors contributed equally to this work.
- Received June 16, 2008.
- Revision received October 29, 2008.
- © 2009 The American Society for Biochemistry and Molecular Biology