Asthma Susceptibility Gene ORMDL3 Promotes Autophagy in Human Bronchial Epithelium

Abstract

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.

Keywords: asthma; autophagy; calcium mobilization; cell death; human bronchial epithelium.

Figure 1.

ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) overexpression promotes autophagy in primary normal human bronchial epithelial (NHBE) and human bronchial epithelial cell line, 16HBE (16HBE14o-) cells. (A and B) Representative electron microscopy (EM) images from two biological replicates show increased number of autophagosomes (white arrows) in 16HBE cells transiently (A) or stably (B) transfected with ORMDL3. Scale bars, 500 nm. (i–iii) Zoom images from B ORMDL3 overexpression group. (C) Blots and protein abundance of autophagy markers measured by Western blot in primary NHBE cells transfected with either empty vector or Flag-tagged human ORMDL3. (D) Western blots were quantified by ImageJ. Means ± SEM are from two to four biological replicates. *P < 0.05, unpaired t test. (E) Confocal images and (F) quantification of autophagosomes by RFP (red fluorescent protein)-GFP-LC3B tandem sensor in 16HBE cells. Yellow and red puncta denote LC3B-positive autophagosomes and autolysosomes, respectively. Scale bar, 20 μm. Means ± SEM shown from 20 image views of control (Ctrl) and 10 image views from each of the two overexpression lines. Cells with five or more puncta or with puncta accumulations are defined as positive. *P < 0.05, unpaired t test. ATG = autophagy related; ATG16L1 = autophagy related 16 like 1.

Figure 2.

ORMDL3 knockdown inhibits autophagy in primary NHBE and 16HBE cells. (A) Detection, and (B) quantification of the autophagy genes by Western blot in primary NHBE cells transfected with anti-ORMDL3 siRNA or negative control (NC). Means ± SEM from three biological replicates with three distinct siRNAs. *P < 0.05, unpaired t test. (C) Representative confocal images of autophagosomes as shown by the RFP-GFP-LC3B tandem sensor in wild-type (WT) and CRISPR-associated protein 9 generated ORMDL3 knockout (KO) 16HBE cells (ORMDL3 KO) treated with or without chloroquine (20 μM) for 16 hours. Scale bar, 20 μm. (D) Quantification of the percentage of autophagy-positive cells at basal levels. *P < 0.05 compared with WT by unpaired t test. Means ± SEM shown from 10 images from each of the two control or KO lines. (E) Representative electron microscopy pictures from two independent experiments in WT or ORMDL3 KO 16HBE stable cells in the presence or absence of chloroquine (20 μM for 16 h). Scale bar, 500 nm. Arrowheads = autophagosomes.

Figure 3.

SERCA2 mediates ORMDL3-induced autophagy. (A) SERCA2 was immunoprecipitated by Flag-tagged ORMDL3 (Flag-IP) in 16HBE cells. Three independent repeats were performed. (B) Representative immunofluorescence from three independent experiments of the expression and colocalization of ORMDL3 (anti-Flag antibody, red) and SERCA2 (anti-SERCA2 antibody, green) in 16HBE cells transfected with Flag-tagged ORMDL3 plasmid. Nuclei were counterstained by DAPI (blue). Scale bars, 20 μm. (C) Detection of autophagy marker LC3B by Western blot in primary NHBE cells treated with SERCA2 inhibitors, thapsigargin (Tg, 10 nM and 100 nM) or cyclopiazonic acid (CPA, 1 μM and 10 μM) for 16 hours. Two independent experiments were performed. (D) 16HBE cells were treated with CPA (20 μM), Tg (100 nM), or chloroquine (CQ) (20 μM) for 20 hours. RNA levels were detected by qRT-PCR experiments. Mean ± SD shown for three independent experiments. *P < 0.05 and **P < 0.01. Statistical methods: ordinary one-way ANOVA and Dunnett’s multiple comparisons. (E) Representative Western blot images and (F) quantification of autophagy gene expression after overexpressing ORMDL3 with or without SERCA2a/SERCA2b co-overexpression in primary NHBE cells. Means ± SEM shown are from four biological replicates per condition. *P < 0.05 compared with control by one-way ANOVA followed by unpaired t test.

Figure 4.

ORMDL3 modulates concentration of intracellular calcium [Ca²⁺]_i mobilization by inhibiting SERCA2 in 16HBE cells. (A) Fluo-4 dye fluorescence signals indicating calcium impulse recorded in 16HBE cells before and after treatment with ATP (10 μM). (B) Representative time-series recordings of Fluo-4 AM fluorescence intensities of calcium concentration in WT 16HBE cells after ATP stimulation. (C) Cytosolic Ca²⁺ decay plots in Ctrl (black) and ORMDL3-overexpressing (ORMDL3, red) 16HBE cells. Means ± SEM from seven biological repeats performed per group. Lines denote the time-dependent recovery of the mean fluorescence intensity expressed as the percentage of peak cytosolic Ca²⁺ intensity. (D) Bar graphs of time to cytosolic Ca²⁺ clearance corresponding to C. (E) Cytosolic Ca²⁺ decay plots in WT (black), ORMDL3 KO (blue) 16HBE cells, and ORMDL3 KO transfected with SERCA2 siRNA (KO + siSERCA2, purple). Means ± SEM from four to nine biological repeats. (F) Seconds needed for cytosolic Ca²⁺ clearance in 16HBE WT, ORMDL3 KO, and ORMDL3 KO transfected with SERCA2 siRNA corresponding to E. *P < 0.05. One-way ANOVA followed by unpaired t test was used for statistical analysis.

Figure 5.

ORMDL3 regulates the cell viability of 16HBE cells. (A) The relative fold change of lactate dehydrogenase (LDH) release in ORMDL3-overexpressing (ORMDL3) cells compared with Ctrl 16HBE lines in the presence or absence of SERCA activator CDN1163 (1 μM) for 16 hours. Means ± SD were from three biological repeats with six technical repeats for each group. (B) The relative fold change of LDH in ORMDL3-deficient (KO) and WT 16HBE cells in the presence or absence of SERCA2 inhibitors CPA (20 μM) or Tg (20 nM) for 16 hours. Means ± SD were from two biological repeats, six technical repeats for each group. *P < 0.05. One-way ANOVA followed by unpaired t test was used for statistical analysis. (C) Apoptosis markers including cleaved Caspase 3 and cleaved PARP were detected in 16HBE cells with various treatments or transfections. Lanes 1 and 4 are from cells treated with etoposide (ETO, 10 μM, 24 h) and TRAIL (100 nM, 6 h), respectively, two inducers of apoptosis, as positive controls. Lanes 2 and 3 are control and ORMDL3-overexpressing lines, respectively. Two independent experiments were performed.

Figure 6.

The correlation of ORMDL3 and autophagy gene expression in human primary bronchial epithelial cells from brushing samples. Scatterplots of linear correlation between expression of ORMDL3 gene and autophagy-related genes—(A) ATG7, (B) ATG12, and (C) ATG16L1—detected in microarray from bronchial epithelial cells obtained from bronchial brushing samples from 44 individuals (27 patients with asthma and 17 control subjects). 95% confidence intervals of correlation are shown in gray.