Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death

Abstract

Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.

Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.

Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.

Measurements and main results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha.

Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.

Figure 1.

Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA)2 protein and RNA expression in non-cystic fibrosis (CF) and CF cell lines. Lysates from cultures of 16HBEo-, CF41o-, and CF45o- cells were analyzed for SERCA2 protein and RNA expression using Western and Northern blot as indicated in Methods. (A and B) Representative Western (experiments repeated six times) and Northern blots (experiments repeated three times) are shown, respectively. To localize SERCA2, cells grown on glass coverslips were fixed and coimmunostained for SERCA2 (red) and the endoplasmic reticulum (ER)-specific protein protein disulphide isomerase (PDI, green) (C). The figure represents one data set from an experiment performed in duplicate. The individual experiment was repeated three times.

Figure 2.

Estimation of endoplasmic reticulum (ER) content (A) and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) (protein and activity) in purified microsomal membranes (B). Live cells cultured in chambered coverglass were stained using ER-Tracker Blue-White DPX (Molecular Probes) (A). The lower panel shows the quantification of fluorescence intensity per whole cell area. For each of three cell lines, about 20 cells were analyzed. Results show means of the data. *Significant difference (P < 0.05) from non-cystic fibrosis (CF) 16HBEo- cells (n = 3). (B, top panel) Representative result of SERCA2 expression in purified microsomal membranes of 16HBEo- (lane 1), CF41o- (lane 2), and CF45o- (lane 3) cells. Microsomal extracts (20 μg) were loaded on 7.5% polyacrylamide gel, and Western blot analysis was performed for SERCA2 expression. (B, lower panel) Thapsigargin (2 μM)-sensitive Ca²⁺ATPase activity in microsomal membranes of normal and CF cells. *Significant difference from 16HBEo- cells (P < 0.05) (n = 3; represents three individual experiments).

Figure 3.

Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) expression in air–liquid interface (ALI), 17–42 days of cultures of primary non-cystic fibrosis (CF) and CF airway epithelial cells. (A) Representative images of in situ immunohistochemistry for SERCA2 expression in cultures fixed with 4% paraformaldehyde (PFA) and stained with 3,3-diaminobenzidine and H₂O₂ for detection of horeseradish peroxidase–coupled mouse secondary antibody. Data shown are from cells from four individual (–4) non-CF donors and four individual (–8) CF donors (one of three separate experiments). Identical staining conditions were used for staining non-CF and CF sections. For Western blot, lysates from the ALI cultures of the cells from the above donors were analyzed by SDS PAGE on 4 to 15% gradient gels, and the proteins were transferred electrophoretically onto nitrocellulose membranes. The membranes were probed with the SERCA2 antibodies at a 1/1,000 dilution (B). (C) Quantification of Western blots for SERCA2 expression in ALI cultures of cells from non-CF and CF donors (14 non-CF and 8 CF) analyzed in three separate experiments. The mean of the non-CF group is the control value; > 60% of CF donors were F508 double mutant, and ∼ 90% had F508 mutation for one allele in the CFTR gene. The bars represent means of data. * Significant difference (P < 0.05) from non-CF cells (results of three individual experiments).

Figure 4.

Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) expression in the epithelium of proximal and distal cystic fibrosis (CF) and non-CF airways. Immunohistochemical localization of SERCA2 was performed as described in Methods using identical conditions for non-CF and CF tissue. Left panel: Nonspecific IgG control. Right panel: SERCA2 staining in epithelium (arrowheads). SERCA2 staining was found predominantly in the epithelium of non-CF bronchi (n = 5 donors) (A) and bronchioles (C), and it was significantly less intense in the epithelium of CF airways (n = 5 donors) (B and D). (E) Quantitation of SERCA2 staining (SERCA2-IgG) in the non-CF and CF bronchi. The regions containing mucus were excluded during quantitation. For each tissue, two SERCA2 and two IgG-stained sections were analyzed, and 10 nonmucus areas per section were randomly selected for quantitation using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD). Similarly, SERCA2 staining in the non-CF and CF bronchioles was quantified (F).

Figure 5.

Expression of low-affinity sarcoendoplasmic reticulum calcium ATPase (SERCA) isoform SERCA3 in cystic fibrosis (CF) and non-CF airway epithelial cells. (A) Western blot for SERCA3 from whole cell lysates (20 μg of protein/lane) from non-CF and CF bronchial airway epithelial cell lines. Membranes were also probed for β-actin to verify equal loading of protein. (B) Northern blot for SERCA3 mRNA expression. About 15 μg of RNA was subjected to Northern blot analysis, and SERCA3 was identified using ³²P-labeled cDNA probe. Before hybridization membranes were analyzed with ultraviolet light exposure for visualization of 18S and 28S RNA bands to verify equality of RNA loading and transfer. Equal loading was further established by 28S RNA (bottom panel) analysis. Cell lysates from air-liquid interface cultures of primary airway epithelial cells from three non-CF (–3) and three CF (–6) subjects were also assessed for SERCA3 expression by Western blot (C). Results represent three individual experiments.

Figure 6.

Effect of cystic fibrosis transmembrane regulator gene inhibitor (CFTR)_inh172 on sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) protein expression. Primary bronchial epithelial cells were cultured for short-term duration on collagen-coated inserts and then treated with 20 μM CFTR_inh172 for 24 hours. Cell lysates were prepared, and Western blot analysis was performed (20 μg protein loaded). (A) SERCA2 expression in CFTR_inh172–treated primary bronchial epithelial cells (lanes 1–3 are untreated control; lanes 4–6 are CFTR_inh172–treated cells). (B) Quantitative data for SERCA2 expression with and without CFTR_inh172 treatment. The bars represent means of data. *Significant difference (P < 0.05) from non-CF cells (results of three individual experiments are shown). (C) Representative blot showing the effect of CFTR_inh172 on SERCA2 expression treatment in CF IB3–1 cells and CFTR-corrected C-38 cells. (D) Quantitative data. Bars represent means of data. *Significant difference (P < 0.05) from untreated cells; n = 3 (experiment repeated twice).

Figure 7.

Effect of inhibiting functional cystic fibrosis transmembrane regulator gene (CFTR) expression by antisense CFTR oligonucleotides (A) or by overexpressing mutated CFTR (B) on sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA)2 expression. Cell lysates were prepared from polarized cultures of 16HBEo- cell line stably transfected with sense (S) and antisense (AS) CFTR oligonucleotide and analyzed for SERCA2 expression by Western blot. The bars represent means of data. *Significant difference (P < 0.05) from control (16HBE-S) cells. The experiment was repeated four times. (B) Results of SERCA2 expression analysis in lysates obtained from control and adenovirally transduced minimally transformed primary human bronchial epithelial cells (UNCN3T) grown on collagen-coated culture dishes. The bars represent means of data. *Significant difference (P < 0.05) from control (LacZ) cells. The experiment was repeated three times (n = 3 per condition).

Figure 8.

Translocation of sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA2) within caveolae-related domains (CRDs) from the estimation of endoplasmic reticulum (ER) of cystic fibrosis (CF) cells. Purified microsomes from 16HBEo- and CF45o- cells were lysed in ice-cold 0.5 M sodium carbonate buffer. The homogenate was adjusted to 45% (w/v) sucrose by the addition of 90% sucrose in the MBS buffer and placed in the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient was established by overlaying this solution with 4 ml of 38% sucrose and 3 ml of 5% sucrose. The tubes were then centrifuged at 4°C for 16 to 18 hours at 130,000 × g, and fractions were manually collected from the top of the gradient. To determine the distribution of CRD-associated proteins within the gradient, each fraction was analyzed by SDS-PAGE, followed by Western blot analysis with SERCA2 and caveolin antibody (A). The Western blot shown is representative of findings in three separate sucrose gradient centrifugation/Western blot experiments. (B and C) Western blot of SERCA2 and Bcl-2 using immunoprecipitate of microsomal fractions from (1) 16HBEo- and (2) CF45o- using Bcl-2 antibody.

Figure 9.

Increased Bcl-2 expression in the cellular compartments of cystic fibrosis (CF) cells. Nuclear, endoplasmic reticulum (ER), and mitochondrial fractions were prepared from (1) 16HBEo-, (2) CF41o-, and (3) CF45o- cells. Western blots were performed using antibodies against Bcl-2, cytochrome c oxidase (mitochondrial marker), protein disulphide isomerase (PDI, ER-specific protein) and lamin C (nuclear marker) (A). (B) Bcl-2 expression in the ER fraction of 16HBEo- cell line stably transfected with sense (S) and antisense (AS) CFTR oligonucleotide. (C) Total Bcl-2 content in cellular lysates of control and adenovirally transduced primary human bronchial epithelial cells grown on collagen-coated culture dishes. The bars represent means of data of two individual experiments (n = 4). *Significant difference (P < 0.05) from control.

Figure 10.

Sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA2) is essential for cell survival during oxidative stress. (A) SERCA2 knockdown using SERCA2 siRNA. Top: representative Western blot of SERCA2 expression in primary human bronchial epithelial cells (grown on collagen-coated culture dishes) (1) mock-transfected or transfected with (2) control or (3) SERCA2 siRNA. Bottom: Quantitation of SERCA2 knockdown. The bars represent means of data. *Significant difference (P < 0.05) from control siRNA-transfected cells (n = 3; three individual experiments). For assessing ozone toxicity, primary human airway epithelial cells were transfected either with a control or SERCA2 siRNA for 24 hours and exposed to 0 ppb or 200 ppb ozone for 18 hours. After exposure, cells were analyzed for cell death using Vybrant apoptosis assay kit. The Alexa Fluor-488–labeled annexin-positive apoptotic and Sytox green-positive dead cells were quantified by flow cytometry (B). The columns represent means of data. *Significant difference (P < 0.05) from 0 ppb control cells. ^#Significant difference from 200 ppb control cells (n = 3). This experiment was repeated four times.