Activation of the unfolded protein response by deltaF508 CFTR

Abstract

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 deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 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 deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 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 deltaF508 CFTR expressed within the same cell.

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 ΔF508 (CFPAC-1, CFBE ΔF508, HeLa ΔF508) 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 ΔF508 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 (C_T) 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 ΔF508 CFTR levels. CFTR_t = relative CFTR mRNA levels measured by the commercial assay; CFTR_WT = relative WT CFTR mRNA levels measured by the MGB probe; CFTR_ΔF508 = relative ΔF508 CFTR mRNA levels calculated from CFTR_t and CFTR_WT; a = ratio of fluorescence signal produced by the MGB probe to the signal produced by the commercial probe.

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 ΔF508 (CFPAC-1, CFBE ΔF508, HeLa ΔF508) 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 ΔF508 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 (C_T) 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 ΔF508 CFTR levels. CFTR_t = relative CFTR mRNA levels measured by the commercial assay; CFTR_WT = relative WT CFTR mRNA levels measured by the MGB probe; CFTR_ΔF508 = relative ΔF508 CFTR mRNA levels calculated from CFTR_t and CFTR_WT; a = ratio of fluorescence signal produced by the MGB probe to the signal produced by the commercial probe.

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 ΔF508 (CFPAC-1, CFBE ΔF508, HeLa ΔF508) 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 ΔF508 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 (C_T) 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 ΔF508 CFTR levels. CFTR_t = relative CFTR mRNA levels measured by the commercial assay; CFTR_WT = relative WT CFTR mRNA levels measured by the MGB probe; CFTR_ΔF508 = relative ΔF508 CFTR mRNA levels calculated from CFTR_t and CFTR_WT; a = ratio of fluorescence signal produced by the MGB probe to the signal produced by the commercial probe.

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 ΔF508 (CFPAC-1, CFBE ΔF508, HeLa ΔF508) 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 ΔF508 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 (C_T) 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 ΔF508 CFTR levels. CFTR_t = relative CFTR mRNA levels measured by the commercial assay; CFTR_WT = relative WT CFTR mRNA levels measured by the MGB probe; CFTR_ΔF508 = relative ΔF508 CFTR mRNA levels calculated from CFTR_t and CFTR_WT; a = ratio of fluorescence signal produced by the MGB probe to the signal produced by the commercial probe.

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.

Figure 3.

Characterization of Calu-3ΔF cells. (A) Relative total CFTR mRNA levels. Total CFTR mRNA levels in Calu-3, Calu-3ΔFC1, Calu-3ΔF, and Calu-3ΔFC5 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 ΔF508 (recombinant) CFTR. Recombinant CFTR mRNA levels were calculated from total (recombinant ΔF508 and endogenous WT) and endogenous CFTR mRNA. Results are plotted as relative distribution of WT and ΔF508 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 (–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-3ΔF 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).

Figure 3.

Characterization of Calu-3ΔF cells. (A) Relative total CFTR mRNA levels. Total CFTR mRNA levels in Calu-3, Calu-3ΔFC1, Calu-3ΔF, and Calu-3ΔFC5 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 ΔF508 (recombinant) CFTR. Recombinant CFTR mRNA levels were calculated from total (recombinant ΔF508 and endogenous WT) and endogenous CFTR mRNA. Results are plotted as relative distribution of WT and ΔF508 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 (–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-3ΔF 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).

Figure 4.

The UPR is constitutively activated in Calu-3ΔFC5. To monitor UPR activity under basal conditions, sXBP1 and BiP mRNA levels were measured in Calu-3, Calu-3ΔFC1, Calu-3ΔFC3, Calu-3ΔFC5, 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-3ΔFC5. Results are plotted relative to GAPDH mRNA. n = 6, *P < 0.001.

Figure 5.

Induction of recombinant ΔF508 CFTR expression and activation of the UPR in Calu-3ΔFC3. Calu-3ΔFC3 cells were treated with NaBu (1 or 2 mM for 12 h) to induce recombinant ΔF508 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-3ΔFC3 cells. Calu-3ΔFC3 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-3ΔFC3 cells. (C) Relative TR mRNA levels in Calu-3ΔFC3 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-3ΔFC3 following NaBu treatment. CFTR was immunoprecipitated from 500 μg total cellular protein using anti-CFTR C-terminal monoclonal antibody (–1), in vitro phosphorylated using ³²P-ATP and PKA, separated on 6% PAGE, and detected by phosphorimaging.

Figure 5.

Induction of recombinant ΔF508 CFTR expression and activation of the UPR in Calu-3ΔFC3. Calu-3ΔFC3 cells were treated with NaBu (1 or 2 mM for 12 h) to induce recombinant ΔF508 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-3ΔFC3 cells. Calu-3ΔFC3 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-3ΔFC3 cells. (C) Relative TR mRNA levels in Calu-3ΔFC3 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-3ΔFC3 following NaBu treatment. CFTR was immunoprecipitated from 500 μg total cellular protein using anti-CFTR C-terminal monoclonal antibody (–1), in vitro phosphorylated using ³²P-ATP and PKA, separated on 6% PAGE, and detected by phosphorimaging.

Figure 6.

Activation of the UPR in Calu-3, Calu-3ΔFC1, Calu-3ΔFC3, Calu-3ΔFC5, 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-3ΔFC3 and Calu-3ΔFC5 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.

Figure 6.

Activation of the UPR in Calu-3, Calu-3ΔFC1, Calu-3ΔFC3, Calu-3ΔFC5, 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-3ΔFC3 and Calu-3ΔFC5 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.