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Published ahead of print on May 5, 2008, doi:10.1165/rcmb.2008-0065OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 448-457, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2008-0065OC

Activation of the Unfolded Protein Response by {Delta}F508 CFTR

Rafal Bartoszewski1, Andras Rab1,2, Asta Jurkuvenaite1,2, Marina Mazur2, John Wakefield3, James F. Collawn1,2 and Zsuzsa Bebok1,2

1 Department of Cell Biology, and 2 Cystic Fibrosis Research Center, University of Alabama at Birmingham, and 3 Tranzyme Inc., Birmingham, Alabama

Correspondence and requests for reprints should be addressed to Zsuzsa Bebok, MD, Department of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd., MCLM 760, Birmingham, AL 35294-0005. E-mail: bebok{at}uab.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient {Delta}F508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3{Delta}F cells that express different levels of endogenous wild-type (WT) and recombinant {Delta}F508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of {Delta}F508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and {Delta}F508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate {Delta}F508 CFTR mRNA levels. The results indicate that overexpression of {Delta}F508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and {Delta}F508 CFTR expressed within the same cell.

Key Words: endoplasmic reticulum stress • unfolded protein response • {Delta}F508 CFTR • quantitative PCR • Calu-3



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Chronic inflammation may cause endoplasmic reticulum (ER) stress and activate the unfolded protein response. Studies regarding ER stress responses are important to understand the pathomechanism of these disorders.

 
Endoplasmic reticulum (ER) stress is often induced by misfolded mutant proteins (1, 2). Cystic fibrosis (CF) caused by the deletion of phenylalanine at the 508 position ({Delta}F508) of the human CF transmembrane conductance regulator (CFTR) is a protein folding disease. This common type of CF is representative of a class of protein folding diseases in which a misfolded, but partially functional, mutant protein is actively degraded by the proteasome (3, 4). Because {Delta}F508 CFTR is functional, large-scale efforts have been directed toward identification of compounds (chemical chaperones, correctors) that rescue this mutant CFTR from endoplasmic reticulum–associated degradation (ERAD). To further these efforts, it is essential to better understand not only how different levels of {Delta}F508 CFTR affect ER function, but also how ER stress responses might regulate CFTR expression.

Previous studies suggested that neither {Delta}F508 CFTR expressed in primary airway cells (5), or introduced by adenovirus gene transfer (6), activated the unfolded protein response (UPR). However, these studies did not examine different expression levels of {Delta}F508 CFTR. Moreover, a recent study revealed that the UPR may be triggered in CF by undefined insults exacerbating the disease (7). This finding also supported earlier observations that activation of the UPR inhibited endogenous CFTR expression (8). It is also conceivable that relatively high {Delta}F508 CFTR protein levels are necessary for its efficient rescue from ERAD. However, it is not known how the increased {Delta}F508 CFTR expression might affect ER functions and the UPR. Importantly, several previously described approaches for rescuing {Delta}F508 CFTR from ERAD (proteasome inhibition [9], ER Ca2+ transport blockers [10, 11], and depletion of ER Ca2+ [12]) also cause ER stress and activate the UPR (13–15).

Based on the idea that the UPR may be a critical component of CF and that emerging treatment approaches such as {Delta}F508 CFTR correction may in fact initiate the UPR, we investigated how increasing the expression of {Delta}F508 CFTR induced ER stress and activated the UPR. We also tested how the UPR affected endogenous and recombinant CFTR expression. To perform these studies, we developed a TaqMan minor groove binding (MGB) based assay aimed at the {Delta}F508 mutation site, for differential measurements of WT and {Delta}F508 CFTR mRNAs expressed in the same cell. Furthermore, we introduce novel epithelial cell line models expressing different levels of both WT and {Delta}F508 CFTR.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
In Vitro Transcription
CFTR transcripts were synthesized from Xhol-cut pcDNA-3.1-WT or pcDNA3.1-{Delta}F plasmids using MEGAscript T7 (Cat # AM1333; Ambion, Austin, TX). The reaction products were purified using MEGAclear (Ambion) according to the manufacturer's recommendations. The control transcript was obtained from the DNA supplied in the kit. RNA concentrations were established by absorbance at 260 nm (A260nm).

Quantification of CFTR mRNA Using Real-Time RT-PCR
Isolation of cellular RNA. Total cellular RNA was isolated using RNeasy (Qiagen, Valencia, CA), according to the manufacturer's recommendations. RNA concentration was calculated based on the absorbance at 260 nm. RNA samples were stored at –20°C.

Measurement of relative mRNA levels using real time RT-PCR. Real-time RT-PCR to measure WT and {Delta}F508 CFTR mRNA (total CFTR, standard assay) was performed using TaqMan One-Step RT-PCR Master Mix Reagents (Cat. No. 4309169; Applied Biosystems, Austin, TX) as described previously (8), using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR System; 2004). GAPDH mRNA and 18S rRNA were amplified as internal controls and used as a reference, as specified for each experiment. WT CFTR mRNA levels were measured using a custom assay (Assay ID: 4331348, WT-CFTR-frag.txt) as described in RESULTS. Forward primer (WT-CFTR frag.txt-73F): GCCTGGCACCATTAAAGAAAAT. Reverse primer (WT CFTR frag.txt-145R): TTTGATGACGCTTCTGTATCTATATTCA. TaqMan minor groove binding (MGB) probe (WT CFTR frag.txt-96T): TCATCTTTGGTGTTTC. The MGB probe recognizes the presence of the TTT that encodes the phenylalaline at the 508 position in human WT CFTR.

Quantification of In Vitro Transcribed CFTR mRNA and the Assessment of Assay Sensitivity Using the MGB-Based Assay
Real-time RT-PCR was performed using TaqMan One-Step RT-PCR Master Mix Reagents and the custom probes containing the MGB probe, as described above. Standard mRNA concentrations were measured based on absorbance at 260 nm (A260nm). Each point represents the average of six replicates.

Quantification of UPR Reporter and Control mRNA Levels
HSPA5/BiP (assay ID: Hs00607129_gH) and spliced XBP1 (assay ID: Hs00231936_m1) mRNA levels were measured to test for UPR activity, as described previously (8). Transferrin receptor (TR, [assay ID: Hs99999911_m1]) was amplified and measured as an additional control.

Cell Lines and Culture Conditions
Calu-3 (16), CFPAC-1 (expressing endogenous {Delta}F508 CFTR) (17), and HeLa cells were obtained from ATCC (Manassas, VA). HelaWT, Hela{Delta}F (8, 18), and Calu-3{Delta}F (expressing recombinant {Delta}F508 CFTR on the Calu-3 background) were transduced with a TranzVector (Tranzyme, Birmingham, AL) and selected as previously described (19). Briefly, {Delta}F508 CFTR cDNA was stably introduced into Calu-3 cells using TranzVector (Tranzyme). TranzVector system represents an HIV-based lentiviral vector with unique safety features as described (18). To generate vector stock, CFTR cDNA was first cloned into the gene transfer component under the control of the human cytomegalovirus (hCMV) promoter. Expression of CFTR was also coupled to the puromycin-N-acetyltransferase gene (puro) gene via the internal ribosomal entry site (IRES) of encephalomyocarditis virus, allowing for rapid selection of cells expressing CFTR in media containing puromycin. Calu-3 cells were transduced at multiplicity of infection of 1 followed by puromycin (4 µg/ml) selection. Puromycin-resistant cells were expanded to form a pool of stable CFTR expressers and subjected to cloning.

Cloning of Calu-3{Delta}F Cells Expressing Endogenous WT and Recombinant {Delta}F508 CFTR
A number of the individual Calu-3{Delta}F clones described above were characterized and established based on the ratio of endogenous (WT) to recombinant ({Delta}F508) CFTR mRNA and protein levels. Clones were divided into three classifications based on whether they expressed (1) low levels of WT and {Delta}F508 CFTR, (2) similar amounts of both recombinant ({Delta}F508) and endogenous (WT) CFTR, and (3) significantly higher levels of recombinant ({Delta}F508) CFTR than endogenous WT. These clones were identified, expanded, and characterized according to CFTR mRNA distribution, protein levels, and morphology.

Antibodies
Anti-CFTR (24–1) C-terminal monoclonal antibody was purified from hybridoma supernatant at the UAB Hybridoma Core Facility (ATCC# HB-11947). Anti-ZO1 rabbit polyclonal antibody was purchased from Invitrogen (Carlsbad, CA). Anti-Sec61β rabbit polyclonal antibody was purchased from Upstate (Temecula, CA).

Induction of ER Stress and Activation of the UPR
Pharmacologic induction of ER stress and activation of the UPR was performed according to previously described methods (8, 15, 20). Briefly, cells were treated with 50 µM ALLN (Sigma, St. Louis, MO) for the time intervals specified in each experiment (15). Tunicamycin (TM) (Sigma) was added to samples for the time periods specified at 5 µg/ml final concentration (20).

Measurement of CFTR Protein Levels
CFTR was immunoprecipitated from 250 µg total protein, in vitro phosphorylated with {gamma}32P ATP (NEN) and cAMP-dependent protein kinase-A (Promega, Madison, WI), separated on 6% gels, and analyzed using phosphorimaging, as described previously (8, 21).

Immunocytochemistry
Cells were grown on 12-mm polycarbonate cell culture inserts (Costar, St. Louis, MO) or on coverslips. Indirect immunocytochemistry and digital imaging were performed as described previously (19, 21).

Induction of Recombinant {Delta}F508 CFTR Expression
Sodium butyrate (NaBu, 1 or 2 mM) was added to cells for 12 hours to induce recombinant CFTR expression, based on previous reports that NaBu induces CFTR expression from a viral promoter (22) but decreases endogenous CFTR expression (23).

Statistical Analysis
Results were expressed as means ± SD. Statistical significance among means was determined using the Student's t test (two samples, paired and unpaired).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Validation of the TTT-Specific MGB Assay as a Method to Specifically Measure WT CFTR mRNA Levels
To confirm the specificity of the MGB-based assay (i.e., its ability to detect and quantitate WT but not {Delta}F508 CFTR mRNA), different cell lines expressing endogenous WT (Calu-3), recombinant WT (HeLaWT), endogenous {Delta}F508 (CFPAC-1), recombinant {Delta}F508 CFTR (HeLa{Delta}F), and cells without CFTR expression (HeLa parental) were tested. After isolation of RNA and reverse transcription, WT CFTR was amplified using custom primers and the TTT-specific MGB probe (see MATERIALS AND METHODS). A short 16-base probe with MGB at the 5' end was used to achieve high specificity in the assay. Average efficiency of this assay was 100% ± 10%. No PCR product was present in samples containing {Delta}F508 CFTR mRNA only (CFPAC-1 and HeLa{Delta}F), confirming the specificity of the assay (Figures 1A and 1B). We also designed a second MGB probe, intended to recognize {Delta}F508. However, assays using this probe did not produce a detectable fluorescent signal during RT-PCR experiments using RNA samples from HeLa{Delta}F or Calu3{Delta}F cells. Therefore, instead of measuring {Delta}F508 CFTR mRNA levels directly, we performed parallel measurements on the same RNA templates, using a standard (commercial) CFTR-specific assay and the MGB-based assay. After determining the difference in the efficiencies of the two assays, we were able to calculate {Delta}F508 CFTR mRNA levels by subtracting WT CFTR mRNA levels (from the MGB-based assay) from total CFTR mRNA levels (from the standard assay).


Figure 1
Figure 1
Figure 1
Figure 1
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  		Figure 1. (A) Specific amplification of wild-type (WT) cystic fibrosis transmembrane conducatnce regulator (CFTR) using the TaqMan MGB-based probe. RNA templates from WT (Calu-3, HeLa WT) and {Delta}F508 (CFPAC-1, CFBE {Delta}F508, HeLa {Delta}F508) CFTR-expressing cells were tested using the custom assay containing the TTT-specific MGB probe in real time RT-PCR experiments. Using the GTT-specific probe, fluorescent signal (PCR product) was only detected in samples obtained from cells expressing WT CFTR. NTC = no template control. (B) Real time RT-PCR amplification of WT CFTR using the MGB-based assay and a commercial probe that amplified both WT and {Delta}F508 CFTR. RNA samples from Calu-3 cells were diluted (log), reverse transcribed, and amplified using either the commercial (A) or the MGB probe (B). NTC = no template control. Increases in reporter dye intensity are shown for the total CFTR probe (A) and the MGB-based probe (B) in the upper panel. Relative efficiency values (CT) are plotted in the lower left (commercial probe) and lower right (MGB probe). (C) Raw fluorescence measurements using Standard TaqMan (A) and MGB (B) probe based assays. Different physical properties of the fluorescent signals generated by standard TaqMan expression (A), and MGB (B) based probes. (D) Formulas to calculate relative {Delta}F508 CFTR levels. CFTRt = relative CFTR mRNA levels measured by the commercial assay; CFTRWT = relative WT CFTR mRNA levels measured by the MGB probe; CFTR{Delta}F508 = relative {Delta}F508 CFTR mRNA levels calculated from CFTRt and CFTRWT; a = ratio of fluorescence signal produced by the MGB probe to the signal produced by the commercial probe.

 
MGB-based assays require either exogenous MGB control or additional calibration to standard TaqMan expression assay for relative quantification. This is necessary because the quality of fluorescent signal generated by the MGB-based product is different than the fluorescent signal generated by the standard assay (Figure 1C). Therefore, for each experimental measurement, we included RNA from parental Calu-3 cells (expressing WT CFTR mRNA only) as a calibration standard. Using this experimental system, we were able to establish the ratio of the fluorescent WT CFTR mRNA signal, generated using the MGB-based assay (WT CFTR), to the fluorescent signal produced by the standard assay (total CFTR). To reduce calculation errors resulting from the different efficiencies of the primers in each sample, four replicates of the calibration samples were conducted for all experiments.

An "a" value (the ratio of the fluorescent signals generated by total [standard] to the WT-CFTR specific [MGB] probes) was calculated for each experiment using samples containing WT CFTR mRNA only (Figure 1D, Formula #1). Subsequently, to calculate amounts of {Delta}F508 CFTR mRNA, we used this "a" value to normalize our experimental measurements from samples containing both WT and {Delta}F508 CFTR mRNA. We could then subtract the amount of WT CFTR mRNA (MGB probe) from the amount of "total" CFTR mRNA (standard assay). The result of this calculation represented the amount of {Delta}F508 CFTR mRNA in the sample (Figure 1D, Formula #2).

Quantification of CFTR mRNA Copy Number Using the MGB-Based Assay
Next, we tested the sensitivity and range of the MGB-based assay using in vitro transcribed WT CFTR mRNA as template. Samples containing known copy numbers of CFTR mRNA were studied. The results indicated that the MGB-based assay was reliable for absolute quantification of WT CFTR mRNA within a wide range, from 106 to as low as 2 to 3 copies (Figure 2), meaning that the assay is suitable for CFTR mRNA measurements in tissue samples or cells with low CFTR expression levels.


Figure 2
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  		Figure 2. Sensitivity of the MGB-based TaqMan probe for WT CFTR. To determine the sensitivity of MGB-based assay to measure WT CFTR mRNA, we performed absolute quantification RT-PCR using known copy numbers of WT CFTR mRNA generated by in vitro transcription reactions. Measurements of CFTR mRNA copies in the range of 104 to 101 are plotted in the upper panel. Low copy number measurements (2–250) are plotted in the lower panel. Each point represents the average of six replicates. R2 values are similar for both high and low copy number measurements.

 
CFTR Expression Levels in the Calu-3{Delta}F Clones
Cloning of Calu-3{Delta}F cells allowed us to select individual clones with different ratios of recombinant {Delta}F508 to endogenous WT CFTR. After expansion of 12 individual clones, we measured their CFTR mRNA and protein levels and calculated their ratios of endogenous WT to recombinant {Delta}F508 CFTR mRNA, as follows: We measured relative total (endogenous WT + recombinant {Delta}F508) CFTR mRNA levels in the clones using the commercial CFTR probe (Figure 3A). We then determined relative WT CFTR mRNA levels using the MGB probe on the same RNA templates (Figure 3B). We used these two measurements to calculate levels of {Delta}F508 CFTR mRNA levels using the method described above. Finally, we calculated the ratios of WT to {Delta}F508 CFTR mRNA using the formula presented in Figure 1C.


Figure 3
Figure 3
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  		Figure 3. Characterization of Calu-3{Delta}F cells. (A) Relative total CFTR mRNA levels. Total CFTR mRNA levels in Calu-3, Calu-3{Delta}FC1, Calu-3{Delta}F, and Calu-3{Delta}FC5 are plotted relative to GAPDH mRNA levels (n = 6). (B) Endogenous CFTR mRNA levels. Endogenous CFTR mRNA levels were measured and plotted relative to GAPDH mRNA (n = 6). (C) Relative distribution of WT (endogenous) and {Delta}F508 (recombinant) CFTR. Recombinant CFTR mRNA levels were calculated from total (recombinant {Delta}F508 and endogenous WT) and endogenous CFTR mRNA. Results are plotted as relative distribution of WT and {Delta}F508 CFTR compared with total CFTR mRNA in parental Calu-3 cells (n = 6). (D) CFTR protein levels. CFTR was immunoprecipitated from 250 µg total cellular protein using anti-CFTR C-terminal monoclonal antibody (24–1), in vitro phosphorylated using 32P-ATP and PKA, separated on 6% PAGE, and detected by phosphorimaging. Arrows show ER (Band B) and post-ER forms (Band C) of CFTR. (E) Immunocytochemical detection of tight junctions, CFTR, and Sec-61 (ER marker) in Calu-3 and Calu-3{Delta}F cells. Cells were grown on permeable supports, and immunocytochemistry was performed on methanol-fixed monolayers. Tight junctions were detected with anti-ZO1 polyclonal antibody (red). CFTR is stained green (24–1 antibody). Images show the apical cell surface at the level of tight junctions (upper panel). CFTR (green) and the ER (Sec-61β, red) were stained to demonstrate CFTR in the ER and post-ER compartments in each clone (lower panel).

 
Clones with similar WT and {Delta}F508 expression levels showed comparable morphologic and physiologic characteristics during the screening process. Therefore, we chose three representative clones (Calu-3{Delta}FC1, Calu-3{Delta}FC3, and Calu-3{Delta}FC5) expressing different ratios of WT to {Delta}F508 CFTR mRNA as models for the present studies (Figure 3C). In Calu-3{Delta}FC1, the ratio of endogenous WT to recombinant {Delta}F508 CFTR mRNA was 1:1, but both mRNA levels were approximately 30% of levels in Calu-3 parental cells. In Calu-3{Delta}FC3, the ratio of endogenous WT to recombinant {Delta}F508 CFTR was also approximately 1:1, but these cells expressed CFTR mRNA levels that were approximately 50% higher than in Calu-3 parental cells. In Calu-3{Delta}FC5, recombinant {Delta}F508 CFTR expression was 8-fold higher than endogenous WT, and endogenous WT CFTR mRNA levels were significantly lower than in Calu-3{Delta}FC3 or Calu-3 parental cells.

Based on their varying mRNA levels, we predicted that CFTR protein levels would also vary between the clones. To confirm this prediction, we used immunoprecipitation and in vitro phosphorylation to quantitavely compare CFTR protein levels in Calu-3, Calu-3{Delta}FC1, Calu-3{Delta}FC3, and Calu-3{Delta}FC5 (Figure 3D). Our results demonstrated that in Calu-3 cells, Band B (core glycosylated, ER form) CFTR was undetectable at steady state, an observation that is consistent with previous results (18) (Figure 3D, lane 1). In contrast, we readily detected Band B CFTR in all three clones (Figure 3D, lanes 2–4). Because no Band B was detected in parental Calu-3 cells, we concluded that the observed Band B in the clones corresponded to recombinant {Delta}F508 CFTR (Figure 3D, Band B). Band C CFTR levels in Calu-3{Delta}FC1 were higher than we expected based on the mRNA analysis. Band C CFTR levels were highest in Calu-3 {Delta}FC3. In Calu-3 {Delta}FC5 cells, Band C CFTR levels were lower than those in parental Calu-3 cells or in the other two clones (Figure 3D, Band C).

We performed immunocytochemistry to investigate potential morphologic differences between clones, compare cell surface CFTR staining, and visualize the ER (Figure 3E). As an ER marker, we probed for Sec-61β, a component of the ER translocon (24). All clones retained the ability to form polarized monolayers, as indicated by the formation of tight junctions (ZO-1 staining, red) (Figure 3E, upper panels). Cell surface CFTR staining is dominant in Calu-3, Calu-3 {Delta}FC1, and Calu-3 {Delta}FC3 cells grown on coverslips (green). In contrast, in Calu-3{Delta}FC5 cells, most of the CFTR fluorescence co-localized with Sec-61β in the ER, and only minimal CFTR staining is present at the cell surface (Figure 3E, lower panels). Since the parental Calu-3 cell line is not clonal, endogenous WT CFTR expression varied between individual cells. This variability may be responsible for the varying levels of endogenous CFTR mRNA in the clones. Another possibility is that the introduction of {Delta}F508 CFTR to Calu-3 cells induced ER stress, activated the UPR, and modulated endogenous CFTR levels (8).

The UPR Is Activated in Calu3{Delta}FC5
To analyze how different levels of {Delta}F508 CFTR affected ER stress and consequent UPR activation, we tested the Calu-3{Delta}F clones, Calu-3 cells, and CFPAC-1 cells for UPR activity. Using two classic reporters of the UPR (relative sXBP1 and BiP mRNA levels) as readouts, we did not detect UPR activity in CFPAC-1, Calu-3{Delta}FC1, or Calu-3{Delta}FC3 cells. In contrast, Calu3{Delta}FC5 cells, which expressed the highest level of recombinant {Delta}F508 CFTR, contained 2-fold higher levels of relative sXBP1 mRNA (P < 0.05; Figure 4A) and 6-fold higher levels of BiP mRNA (P < 0.001, Figure 4B) than the controls. Relative TR mRNA levels, measured as a control, were similar in all of the Calu-3 clones and in CFPAC-1 cells (Figure 4C). These results revealed constitutive UPR activity in Calu-3{Delta}FC5, the clone expressing the highest level of {Delta}F508 CFTR.


Figure 4
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  		Figure 4. The UPR is constitutively activated in Calu-3{Delta}FC5. To monitor UPR activity under basal conditions, sXBP1 and BiP mRNA levels were measured in Calu-3, Calu-3{Delta}FC1, Calu-3{Delta}FC3, Calu-3{Delta}FC5, and CFPAC-1 cells. Transferrin receptor (TR) mRNA was measured as a control and did not change significantly. Constitutive UPR activity was observed only in Calu-3{Delta}FC5. Results are plotted relative to GAPDH mRNA. n = 6, *P < 0.001.

 
Induction of Recombinant {Delta}F508 CFTR Expression in Calu-3{Delta}FC3 Cells Activated the UPR and Decreased Endogenous CFTR mRNA Levels
To clarify the role of {Delta}F508 CFTR on UPR activation, we conducted follow-up experiments to test whether increased levels of {Delta}F508 CFTR in the ER activated the UPR. We also wanted to confirm that the high {Delta}F508 CFTR levels in Calu-3{Delta}FC5 were responsible for the increases in sXBP1 and BiP mRNA levels, For these experiments, we boosted the recombinant CFTR expression in the Calu-3{Delta}FC3 cells (1:1 recombinant to endogenous CFTR expression under unstimulated conditions) and tested for UPR activity. To enhance recombinant CFTR expression, we treated Calu-3{Delta}FC3 cells with NaBu, which induces the expression of CFTR from retroviral vectors (22). In contrast, treatment of these cells with 1 mM NaBu for 12 hours decreased WT CFTR mRNA levels to 20 to 30% (Figure 5A). NaBu treatment of the Calu-3{Delta}FC3 cells also decreased levels of endogenous WT CFTR mRNA, but it increased levels of total CFTR mRNA (Figure 5B). TR mRNA levels, measured as a control, decreased slightly but not significantly in both Calu-3 and Calu-3{Delta}FC3 cells after NaBu treatment (Figure 5C). In both Calu-3 and Calu-3{Delta}FC3 cells, NaBu decreased endogenous CFTR mRNA levels. Therefore, the increased levels of total CFTR mRNA in Calu-3{Delta}FC3 cells must have corresponded to increased levels of recombinant {Delta}F508 CFTR. The significant decrease in endogenous WT and increase in recombinant {Delta}F508 CFTR mRNA levels were accompanied by decreased Band C CFTR levels in Calu-3 and decreased Band C and increased Band B CFTR protein levels in Calu-3{Delta}F cells (Figure 5E). Taken together, these results demonstrated that in Calu-3{Delta}FC3 cells, NaBu treatment enhanced the expression of recombinant {Delta}F508 CFTR at the mRNA and protein levels.


Figure 5
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  		Figure 5. Induction of recombinant {Delta}F508 CFTR expression and activation of the UPR in Calu-3{Delta}FC3. Calu-3{Delta}FC3 cells were treated with NaBu (1 or 2 mM for 12 h) to induce recombinant {Delta}F508 CFTR expression. (A) Relative CFTR mRNA levels in Calu-3 cells. Total and endogenous CFTR mRNA levels were measured in control and NaBu-treated Calu-3 cells. A significant decrease in CFTR mRNA was detected upon NaBu treatment. Results are plotted as relative CFTR mRNA compared with control (untreated) cells. (B) Relative CFTR mRNA levels in Calu-3{Delta}FC3 cells. Calu-3{Delta}FC3 cells express both endogenous and recombinant CFTR. A significant increase in total and a decrease in endogenous CFTR mRNA were observed in NaBu-treated cells compared with control cells. Results are plotted relative to CFTR mRNA levels in control (untreated) Calu-3{Delta}FC3 cells. (C) Relative TR mRNA levels in Calu-3{Delta}FC3 cells. TR mRNA levels were monitored as a control and were slightly decreased after NaBu treatment. TR mRNA levels are plotted relative to 18S rRNA. (D) Relative sXBP1 and BiP mRNA levels. Relative sXBP1 and BiP mRNA levels are plotted as a fold increase after NaBu treatment. Calu-3 parental cells were tested as controls; n = 6, *P < 0.01 (sXBP1); P < 0.005 (BiP). (E) Changes in CFTR protein levels in Calu-3 and Calu-3{Delta}FC3 following NaBu treatment. CFTR was immunoprecipitated from 500 µg total cellular protein using anti-CFTR C-terminal monoclonal antibody (24–1), in vitro phosphorylated using 32P-ATP and PKA, separated on 6% PAGE, and detected by phosphorimaging.

 
We investigated whether we had activated the UPR in these cells by increasing {Delta}F508 CFTR expression. To explore this possibility, we compared the relative sXBP1 and BiP mRNA levels in untreated and NaBu-treated Calu-3{Delta}FC3 cells. We also examined parental Calu-3 cells as control. NaBu-treated Calu-3{Delta}FC3 cells exhibited significantly higher levels of both sXBP1 and BiP mRNA than untreated cells (Figure 5D). We observed no increase in either of these UPR markers after NaBu treatment of parental Calu-3, HeLaWT (18), or Calu-3WT cells (8) transduced with TranzVector encoding WT CFTR (data not shown). Based on these results, we concluded that the NaBu-mediated increase in recombinant {Delta}F508 CFTR expression resulted in ER stress and UPR activation. These results support the idea that, in contrast to what was shown for WT CFTR (8), {Delta}F508 CFTR caused ER stress and activated the UPR when expressed at high levels.

Pharmacologic Induction of ER Stress and Activation of the UPR in Calu-3{Delta}F Cells Decreased Endogenous WT CFTR Expression
To confirm that ER stress and activation of the UPR mediated endogenous, but not recombinant, CFTR mRNA repression, we induced ER stress in Calu-3, Calu-3{Delta}FC1, Calu-3{Delta}FC3, Calu-3{Delta}FC5, and CFPAC-1 cells by two classic pharmacologic methods (tunicamycin or proteasome inhibition) (8, 15). Throughout our studies herein, we obtained similar results using either of these ER stressors. We monitored for UPR activity by following sXBP1 mRNA levels. As expected, induction of ER stress increased sXBP1 mRNA levels in parental Calu-3 cells, all Calu-3{Delta}F clones, and CFPAC-1 cells (Figure 6A). Interestingly, although sXBP1 mRNA levels were constitutively higher in Calu-3{Delta}FC5 cells, pharmacologic ER stress mediated an additional increase.


Figure 6
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  		Figure 6. Activation of the UPR in Calu-3, Calu-3{Delta}FC1, Calu-3{Delta}FC3, Calu-3{Delta}FC5, and CFPAC-1 cells with tunicamycin (TM). (A) sXBP1 mRNA levels were measured to monitor UPR activity after TM treatment. Results are plotted relative to sXBP1 mRNA in Calu-3 parental cells. (B) Relative total (left panels) and endogenous (right panels) CFTR mRNA levels in Calu-3{Delta}FC3 and Calu-3{Delta}FC5 after induction of ER stress and activation of the UPR. ALLN or TM were used to induce ER stress and activate the UPR. Results are plotted relative to CFTR mRNA levels in Calu-3 cells. n = 6, P < 0.005. (C) Induction of ER stress and activation of the UPR decrease endogenous CFTR mRNA levels in CFPAC-1 cells. CFPAC-1 cells were treated with TM to induce ER stress. The UPR was monitored by measuring sXBP1 mRNA (left panel). CFTR mRNA levels were measured using a primer set that amplifies both endogenous and recombinant CFTR (right panel). Results are plotted relative to control (untreated) cells.

 
We also investigated relative total and endogenous CFTR mRNA levels after pharmacologic ER stress (Figure 6B), and our measurements indicated that pharmacologic activation of the UPR decreased endogenous CFTR mRNA levels in parental Calu-3 cells to less than 20% of controls. Subjecting Calu-3{Delta}FC5 cells to the same conditions increased total CFTR mRNA levels, whereas endogenous CFTR mRNA dropped to almost undetectable levels. These observations suggested that despite the existing signs of ER stress in Calu-3{Delta}FC5 under basal conditions, the additional acute increase in UPR activity completely abolished endogenous CFTR expression. We observed no significant changes in relative TR mRNA levels under identical experimental conditions (data not shown).

To elucidate whether ER stress mediated similar effects on endogenous {Delta}F508 CFTR mRNA levels, we included CFPAC-1 cells in these experiments. Although {Delta}F508 CFTR mRNA levels are very low in CFPAC1 cells under basal conditions, activation of the UPR using proteasome inhibition or TM further decreased their endogenous {Delta}F508 CFTR mRNA levels. We measured no significant changes in TR mRNA levels under the same conditions (data not shown). These results from CFPAC-1 cells indicated that the UPR has a pronounced negative effect on endogenous CFTR mRNA, an effect that is detectable even in cells with very low CFTR mRNA expression.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our studies concentrate on membrane protein expression regulation during ER stress. Earlier studies indicated that pharmacologically induced ER stress and the UPR decreased endogenous, but not recombinant, CFTR expression at the mRNA and protein maturational levels, and that overexpression of WT CFTR did not by itself cause ER stress (8). Herein, we showed that {Delta}F508 CFTR is fundamentally different from WT, in that {Delta}F508 CFTR can induce ER stress when expressed at high levels.

The importance of using endogenous CFTR-expressing cell lines (such as Calu-3) as model systems is clear from previous studies (8, 18). However, endogenous {Delta}F508 CFTR–expressing models are limited and the expression level of the mutant CFTR is usually too low to study trafficking. Therefore, we developed and characterized clonal cell lines that express {Delta}F508 CFTR on the WT Calu-3 background. These Calu-3{Delta}F clones stably express different levels of WT and {Delta}F508 CFTR, as defined herein. Together, these cell lines provide a novel model system in which the effects of different WT and {Delta}F508 CFTR expression levels can be explored. In addition, these cells also represent a model system in which endogenous and recombinant species of CFTR exist within the same cells, facilitating a wide range of studies such as the NaBu induction experiments presented herein.

In previous studies, NaBu has been used to facilitate CFTR expression and to enhance the rescue of recombinant {Delta}F508 CFTR mediated by low temperature culture (22, 25, 26). In contrast, other studies showed that in parental Calu-3 cells, NaBu treatment decreased endogenous CFTR function (27) and mRNA levels (23). Our results are in agreement with the latter two studies, in that NaBu increased recombinant CFTR expression but significantly decreased endogenous CFTR mRNA levels.

Because we found that the decrease in endogenous CFTR mRNA levels was not accompanied by UPR activation, and because recombinant CFTR levels increased by more than 5-fold, we used NaBu treatment to test the effect of increased {Delta}F508 CFTR expression on ER stress induction and UPR activation. These experiments confirmed our previous hypothesis that {Delta}F508 CFTR differs from WT in its ability to cause ER stress (8). These results also support previous reports that ER traffic and degradation of WT and {Delta}F508 CFTR are different (28, 29).

Although our studies concentrate on ER stress, and the decrease in CFTR mRNA levels under ER stress results from transcriptional repression (8), it is likely that ER stress occurs in combination with cytosolic stress in vivo. In support of this idea, oxidative stress caused by butylhydroquinone exposure decreased CFTR mRNA stability in a previous report (30). In addition, exposure to cigarette smoke extract, most likely a source of ER and cytosolic stress, decreased chloride secretion in human bronchial epithelial cells (31). Furthermore, inflammatory cytokines decreased CFTR mRNA stability by activating exosomes through the AU-rich element of the 3'UTR (32). Although none of these reports included a study of ER stress and UPR activity under the described conditions, other investigators have reported that infection and inflammation in CF airway epithelia triggered the UPR (33). Therefore, it is feasible that the cumulative negative effects of cellular stress on CFTR expression might down-regulate functional CFTR levels to pathologically low levels, especially considering that epithelial cells often bear the brunt of these stress conditions. These observations are especially relevant with regard to CFTR function in patients with compound heterozygote CF. If our hypotheses are correct, any strain on the cells might disturb the partially balanced CFTR function in these patients, resulting in a suppression of CFTR expression with potential detrimental effects.

Understanding the role of {Delta}F508 CFTR in ER stress and UPR activation is critical to develop therapies for CF caused by the {Delta}F508 mutation. Significant efforts toward identifying {Delta}F508 CFTR rescue compounds have produced some promising candidates (34–39), but the mechanisms by which any of these rescue agents work are not fully understood, and many of them are likely to increase {Delta}F508 CFTR levels in the ER. It is therefore vital to understand how increased levels of the mutant protein may initiate other cellular responses. If any of these compounds causes ER stress, the inhibitory effect on CFTR expression would only be apparent in endogenous CFTR-expressing models. For all of these rescue agents, the initial characterization studies were conducted in stable recombinant {Delta}F508 CFTR-expressing cell lines, and only a few endogenous {Delta}F508-CFTR expressing models have been used (36, 38).

In addition, it is necessary to accurately differentiate between WT and {Delta}508 CFTR mRNA levels when analyzing therapeutic approaches and pathologic influences on CFTR transcript levels or in heterozygote settings. Moreover, differential quantification of WT and {Delta}F508 mutant CFTR transcripts has furthered our understanding of how epigenetic factors influence CFTR expression and function (8, 40). Various studies have introduced a number of PCR- and non–PCR-based methods to investigate CFTR transcript levels (41, 42), but future studies will require novel quantitative, specific, and sensitive assays that are able to discriminate between WT and {Delta}F508 CFTR mRNA. As such, our WT CFTR–specific, MGB-based RT-PCR protocol fits these requirements and will serve as an important tool to study the molecular pathomechanism of CF.

In summary, to clarify how {Delta}F508 CFTR mRNA levels relate to activation of the UPR, we developed novel cell lines and a novel RT-PCR based quantitative assay to differentially investigate WT and {Delta}F508 CFTR mRNA levels. Our findings showed that overexpression of recombinant {Delta}F508 CFTR induced ER stress and activated the UPR. Endogenous CFTR (WT and {Delta}F508) expression decreased under ER stress conditions, independent of the pathways of ER stress induction. Therefore, these studies provide novel insight into how cellular stress responses regulate CFTR expression.


   Footnotes
 
This work was supported by a grant from the National Institutes of Health (HL076587 to Z.B.).

Originally Published in Press as DOI: 10.1165/rcmb.2008-0065OC on May 5, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 8, 2008

Accepted in final form April 8, 2008


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 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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