SERCA2 regulates non-CF and CF airway epithelial cell response to ozone

Abstract

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.

Figure 1. Ozone-induced toxicity in non-CF and CF airway epithelial cells.

Polarized cultures of non-CF (16 HBEo-) and CF (CF41o- and CF45o-) cells were exposed to either 0 or 500 ppb ozone for 8 h. At the end of exposure cells were stained with Calcein AM (green, live) and propidium iodide (PI) (red, dead) (A). Quantitation of dead PI positive cells was performed using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD) and shown in (B). The percentage of dead cells was determined as the percentage of adherent positive cells relative to total number of adherent cells per ten high-power fields (x20). The assessment was performed blindfolded by two different individuals and the data obtained was analyzed by a statistician. The data shown are mean±SEM (n = 3). The image is representative of three independent experiments. * Indicates significant difference from 0 ppb control and # indicates significant difference from 500 ppb exposed non-CF, p<0.05. (C) Polarized cultures of 16HBEo- (-□-) and CF41o- (-▵-) and CF45o- (-○-) cells were exposed to either 0 (open symbols) or 500 ppb (closed symbols) ozone for 2, 4 or 6 h. At the end of each exposure supernatant media was collected and analyzed for caspase 3/7 release as described in the Methods. The data shown are mean±SEM (n = 3). * Indicates significant difference from 0 ppb control, and # indicates significant difference from 500 ppb exposed non-CF p<0.05. (D) Polarized cultures of non-CF 16HBEo- (white columns) and CF CF41o- (black columns) cells were exposed to either 0 or 500 ppb ozone for 8 h following which cells were collected and Annexin binding assay was performed as described in the Methods. Data is from one representative of 2 experiments. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb control and # indicates significant difference from 500 ppb exposed 16HBEo-. Cells p<0.05 (E) 16HBEo- and CF41o- cells were cultured on fibronectin-coated 6-well plates and exposed to ozone on the 4^th day of plating. Measurement of chloromethyltetramethylrosamine (MitoTracker Orange, Molecular Probes) fluorescence, an indicator of mitochondrial membrane potential (MMP), was performed as described in the Methods. A mean fluorescence intensity (MFI) of cells exposed to 0 ppb ozone was taken as control value. The data shown are mean±SEM (n = 6) The image is representative of two independent experiments. * Indicates significant difference from non-CF cells, p<0.05. (F) Ozone-mediated cytochrome c release in non-CF and CF cells. 16HBEo- and CF41o- cells were cultured on glass coverslips in 6-well plates and exposed to ozone (200 ppb) for 4 h. Cells were fixed and processed for cytochrome c releases using immunocytochemistry as described in the text. Representative images of (a) 16HBEo- and (b) CF41o- cells exposed to 200 ppb ozone are shown. The arrows indicate staining of cytochrome c. (G) Western blot of cytochrome c (‘Cyt c’) in the cytosolic fraction of 0 ppb and 200 ppb (4 h) ozone-exposed 16HBE (1), CF41o- (2) and CF45o- (3) cells. Image is representative of three independent experiments. The lower panel is a bar graph showing quantitation of cytochrome c Western bands. The data shown are mean±SEM (n = 3). * Indicates significant difference from 0 ppb control, and # indicates significant difference from 200 ppb (closed bars) exposed non-CF, p<0.05.

Figure 2. Ozone-mediated cytokine release in polarized cultures of non-CF and CF cell lines.

16HBEo- (white column) and CF41o- (black column) cells were cultured on collagen-coated 30 mm inserts. Once cells were polarized (8-10 days in culture) cells were exposed to 0, 100 or 200 ppb ozone with 200 µl media on the apical surface. At the end of exposure (18 h) an additional 200 µl media was added to the apical surface. Aliquots were collected after 4 h and analyzed for IL-8 (A), G-CSF (B) or GM-CSF (C) as described. The data shown are mean±SEM (n = 6). The image is representative of four independent experiments. * Indicates significant difference from 0 ppb control and # indicates significant difference from 100 or 200 ppb exposed non-CF p<0.05.

Figure 3

Effect of ozone exposure on the release of proinflammatory cytokines in differentiated cultures of non-CF (▵, 0 ppb and ▴, 200 ppb) and CF (○, 0 ppb and •, 200 ppb) primary airway epithelial cells. Cells were cultured on collagen-coated snapwells and allowed to grow and differentiate for 30 days. Before ozone exposure 100 µl media was added to the apical surface. At the end of exposure (18 h) an additional 200 µl media was added apically. After 4 h the media from apical (A) and basolateral (B) surface was collected and 100 µl of 6 mM DTT in PBS was added on the apical surface. The cells were incubated for 15 min in DTT and the supernatant fluid was carefully collected using a 1.0 ml pipette tip. The media and DTT solubilized mucin were analyzed for cytokines (IL-8 in A & B, G-CSF in C & D and GM-CSF in E & F) as described in the Methods. For apical cytokine content media and DTT solubilized mucin samples were pooled. Each symbol represents the mean of each donor. Three separate experiments were performed each time using one non-CF (donor 1-3) and one CF (4-6) donor. * Indicates significant difference from 0 ppb exposed non-CF, p<0.05 and # indicates significant difference from 200 ppb non-CF, p<0.05 and $ indicates significant difference from 0 ppb CF, p<0.05.

Figure 4. Lentiviral shRNA-mediated SERCA2 knockdown and its effect on cell phenotype and viability.

A) Primary airway epithelial cells were cultured on collagen-coated 24-well plates. Replication-deficient lentivirus each carrying 5 different SERCA2 target shRNA (pLKO.1-CMV-tGFP-ATP2A2) (SERCA2A-1-5) were transduced at different MOI according to manufacturer's instructions and SERCA2 knock down was tested using real time RTPCR. The data shown are mean±SEM (n = 3) *p<0.05, significant difference between cells treated with SERCA2 shRNA and control. B) Primary airway epithelial cells were cultured on collagen-coated 6-well plates and transduced with SERCA2 shRNA (pLKO.1-CMV-tGFP-ATP2A2) or pLKO.1-CMVtGFP-TurboGFP control. Forty-eight hour post transduction cell lysates were prepared and SERCA2 Western blot was performed. C) Airway epithelial cell phenotype with and GFP expression by control and SERCA2 shRNA transduced cells. Cultured cells retained the morphology of airway epithelial cells (Brightfield light microscopy of untransduced, A & B, transduced C & E) and expressed GFP (Fluorescence microscopy of transduced D & F, matching area to the Brightfield in C & E). D) Flow cytometry of control, SERCA2 shRNA (pLKO.1-CMV-tGFP-ATP2A2) or pLKO.1-CMVtGFP-TurboGFP control cells with 7-AAD a cell death marker.

Figure 5. SERCA2 regulates ozone-mediated cytokine release by primary airway epithelial cells.

Cells were cultured on collagen-coated snapwells at air-liquid interface (ALI) or on collagen-coated 6-well plates. (A) Primary airway epithelial cells were cultured at ALI and exposed to 500 or 1000 ppb ozone. At the end of exposure (4 h), lysates were prepared and SERCA2 protein was analyzed using Western blot. A representative blot from cells of one donor is shown. (B) Cells transduced with lentiviral GFP control or lentiviral GFP-SERCA2 shRNA were cultured on collagen-coated snapwells at air-liquid interface (ALI) and exposed to 0 (white column) or 200 (black column) ppb ozone as described in legend to Figure 3. Apical media was collected and analyzed. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb control and # indicates significant difference from 200 ppb exposed lentiviral GFP transduced cells, and $ indicates significant difference from 0 ppb exposed lentiviral GFP-SERCA2 shRNA transduced cells p<0.05. C) Primary airway epithelial cells cultured on 6-well plates were transduced with Ad.GFP or Ad.SERCA2. Exposure to ozone, 0 (white column) or 200 (black column) ppb for 18 h was carried out 48 h post transduction. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb and # indicates significant difference from 200 ppb ozone-exposed Ad.GFP transduced cells p<0.05.

Figure 6. Ozone-induced cytokine release is NF-κB dependent.

A) Effect of preincubation of cells cultured on collagen coated 6-well plates for 30 min with 10 µM NF-κB inhibitor [6-amino-4- (4-phenoxyphenylethylamino) quinazoline] or diluent on IL-8 release. The data shown are mean±SEM (n = 3). The image is representative of four independent experiments. * Indicates significant difference from 0 ppb exposed cells, # indicates significant difference from non-CF and $ indicates significant difference from CF cells not treated with NF-κB inhibitor during ozone (200 ppb) exposure p<0.05. For analysis of NF-κB activation, nuclear p65 was measured in non-CF and CF cells cultured on collagen-coated 6-well plates as described in the Methods (B). Cells were harvested and nuclear lysates were prepared after exposure to 200 ppb ozone. The nuclear fractions were assessed for p65 using the method described. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 200 ppb ozone-exposed non-CF cells p<0.05.

Figure 7. SERCA2 regulates NF-κB promoter activity and IL-8 release.

NF-κB luciferase reporter-expressing 16HBEo- cells were cultured on collagen-coated 6-well plates and transfected with either control siRNA or SERCA2 siRNA. Forty-eight hour post transfection cells were treated with ‘Cytomix’ containing TNFα (20 ng/ml), IL-1β (10 ng/ml), IFNγ (10 ng/ml) and LPS (50 ng/ml) with or without thapsigargin (thaps; 2 µM)). (A) Cell lysates were prepared 24 h after treatment and luciferase activity was measured as described. The data shown are mean±SEM (n = 6). The image is representative of four independent experiments. * Indicates significant difference from untreated siControl transfected cells, # indicates significant difference from untreated siSERCA2 transfected cells, $ indicates significant difference from cytomix-treated siControl transfected cells, and $$ indicates significant difference from cytomix+thaps-treated siControl-transfected cells p<0.05. (B) Cells were cultured and treated as described above and supernatant media was collected and analyzed for IL-8. The data shown are mean±SEM (n = 3). The image is representative of two independent experiments. * Indicates significant difference from untreated siControl transfected cells, # indicates significant difference from untreated siSERCA2 transfected cells, $ indicates significant difference from cytomix-treated siControl transfected cells and $$ indicates significant difference from cytomix+thaps-treated siControl-transfected cells p<0.05.

Figure 8. SERCA2 modulates ozone-induced NF-κB activity and IL-8 release.

NF-κB luciferase reporter-expressing 16HBEo- cells were cultured on collagen-coated 6-well plates and transfected with either control siRNA or SERCA2 siRNA. Forty-eight hour post transfection cells were exposed to ozone (0 or 500 ppb). (A) Cell lysates were prepared after treatment and luciferase activity was measured as described. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb siControl transfected cells, # indicates significant difference from 500 ppb siControl transfected cells p<0.05. (B) Cells were cultured and treated as described above and supernatant media was collected and analyzed for IL-8. The data shown are mean±SEM (n = 6). The image is representative of two independent experiments. * Indicates significant difference from 0 ppb siControl transfected cells and # indicates significant difference from 500 ppb siControl transfected cells.

Figure 9. SERCA2 overexpression mitigates cytomix-induced enhanced IL-8 release and NF-κB activity in CF cells.

16HBEo- cell lines stably transfected with sense (16HBE-S, non-CF) and antisense (16HBE-AS, CF) CFTR oligonucleotide were cultured on 6-well plates as described in the Methods. On the 2^nd day of plating they were transduced with Ad.GFP or Ad. SERCA2 as described in the Methods. Cells were exposed to cytomix after 24 h incubation. Supernatant media was collected for IL-8 assay (A) and nuclear lysates were prepared for total p65 assay by ELISA. The data shown are mean±SEM (n = 6). * Indicates significant difference from untreated control cells, # indicates significant difference from cytomix treated non-CF and ! indicates significant difference from cytomix treated non-CF and CF cells p<0.05.