Hyperoxia induces LC3B expression in mouse lung. C57BL/6 mice were exposed to room air or to hyperoxia (100% O₂) for the indicated times. (A) The expression of LC3B in total lung homogenates was determined by Western immunoblot analysis. Western blot is representative of n = 2 mice for each exposure time. β-actin served as the standard. Relative LC3B-II levels were quantified by densitometry and normalized to β-actin (n = 5 animals for each group). *P < 0.01. A time course of hyperoxia-induced LC3B was determined by Western immunoblot analysis in human lung epithelial cells (Beas2B) (B) and in primary human bronchial epithelial cells. (C) β-actin served as the standard. (B, C) Figures are representative of at least three independent experiments. Densitometric analyses are shown in the bar graph.

Effect of LC3B in hyperoxia-induced epithelial cell apoptosis. (A) Primary mouse lung epithelial cells were isolated from C57/BL6 mice. Cells were then exposed to room air (RA) or hyperoxia (24 h). Annexin V and PI were used to stain cells, and fluorescence-activated cell sorter analysis was performed. Inset: Cleaved caspase 3 was determined using Western blot analysis. (B) Beas-2B cells were exposed to hyperoxia for up to 48 hours and harvested at the indicated times. Total protein extracts (25 μg) were subjected to immunoblot analysis with antibodies against the cleaved form of caspase-3 (c-caspase-3) or poly-(ADP ribose) polymerase (PARP). β-actin served as the standard. (C) Beas-2B cells were transfected with control vectors or LC3B overexpression clones. After 24 hours, cells were exposed to hyperoxia (24 and 48 h). Hyperoxia-induced cleaved caspase 3 (active form) was determined using Western blot analysis. Overexpression of LC3B was confirmed by protein level determined using Western blot analysis (inset). All results represent three independent experiments.

Crosstalk of autophagy with apoptosis. (A) Beas-2B cells were transfected with control siRNA or LC3B siRNA. After 24 hours, cells were exposed to hyperoxia (4 h). Co-IP assays were performed to detect the interaction between Fas and caspase 8. (B) Beas-2B cells were transfected with control vector or LC3B expression vector. After 24 hours of transfection, cells were exposed to hyperoxia (4 h). Co-IP assays were performed to detect the interaction between Fas and caspase 8. (C) Beas2B cells were exposed to normoxia or hyperoxia for 4 hours (upper panels) and 24 hours (lower panels). The interaction between Fas and LC3B was determined by co-IP assays. (D) Beas2B cells were exposed to normoxia or hyperoxia for 4 hours. Lipid rafts were isolated, and the amount of LC3B trafficking into the lipid raft fraction was determined using Western blot analysis. (E) Beas2B cells were transfected with caveolin-1 wild-type Y14 clone and Y14D (tyrosine to aspartate) mutant clones. Cells were then exposed to hyperoxia (24 or 48 h). LC3B and cav-1 interaction was determined using co-IP assays and Western blot analysis. NS = nonspecific binding. Results are representative of three independent experiments.

Hyperoxia induces autophagy in cultured epithelial cells. Beas-2B cells were transfected with 20 μg of GFP-LC3B using Lipofectamine TM2000 (Invitrogen, Carlsbad, CA) in OPTI-MEM and then exposed to normoxia or hyperoxia for 24 hours. The cells were observed under fluorescent microscopy. (A) Representative image is shown (original magnification: ×40). (B) Percentages of cells with punctuated GFP-LC3 were quantified. Results are expressed as means ± SEM of more than 100 cells per sample. Similar results were obtained from three independent experiments. *P < 0.01 compared with control. (C) The representative electron micrograph shows the ultrastructure of Beas-2B cells exposed to standard cultured conditions (normoxia) or hyperoxia for 24 hours. Representative morphology of autophagic vacuoles is shown, with evidence of autophagosome formation. Nu = nucleus; white arrows, autophagic vacuoles. Scale bar, 2 μM. (D) The average number of autophagic vacuoles was determined from randomly selected samples of more than 30 cells. The total numbers of autophagosomes per cell are expressed as means ± SEM. *P < 0.01 compared with control.

Involvement of c-Jun N-terminal kinase (JNK) in hyperoxia-induced autophagy. (A) Beas2B cells were exposed to normoxia and hyperoxia (12 h). mRNA was isolated and reverse transcribed, and Taqman real-time PCR was performed using LC3B primer and probe. Relative folds of increased mRNA transcription were displayed. (B) Total protein extracts (25 μg) were obtained from Beas-2B cells exposed to hyperoxia for 12 hours in the presence or absence of SP600125 (5 μM) and were subjected to immunoblot analysis with antibodies against LC3B, JNK, and phospho-JNK (pJNK). (C) Beas-2B cells or HBE cells were exposed to normoxia or hyperoxia for 24 hours in the presence or absence of N-acetyl-L-cysteine (NAC) (5 mM), diphenyleneiodonium (DPI) (10 μM), and mito-TEMPO (100 μM). Immunoblot analyses were performed to determine the levels of LC3B, and β-actin served as the standard. All results represent three independent experiments.

Cellular function of LC3B in hyperoxia-induced cell death. (A) Beas-2B cells were transfected with control-siRNA and LC3B-siRNA for 30 hours followed by exposure to normoxia or hyperoxia for 48 hours. Cells were labeled with FITC-Annexin V and propidium iodide (PI) and analyzed with flow cytometry (n = 7). Annexin V(−) and PI(−) cells were defined as live cells. The expression of LC3B was determined by Western immunoblot analysis. β-actin served as the standard (inset). (B) Cells were treated as in (A). Lactate dehydrogenase (LDH) levels in the supernatant were measured by Cytotoxicity Detection Kit (Roche Applied Science, Indianapolis, IN) (n = 8). Results are expressed as means ± SEM of indicated numbers of independent experiments. *P < 0.05 compared with indicated groups. Statistical significance was determined by ANOVA with multiple comparisons. (C) Primary lung fibroblasts were harvested from wild-type and LC3B^+/− mice. Equal numbers of cells (1 × 10⁶ cells per dish) were cultured on the 60-mm dishes and exposed to hyperoxia. Cells were harvested at 24 and 48 hours. (Upper panel) Total protein extracts (25 μg) were subjected to Western immunoblot analysis with antibodies against LC3B. (Lower panel) LDH levels in the supernatant were measured by Cytotoxicity Detection Kit. Results are expressed as means ± SEM of five independent experiments. *P < 0.05 and **P < 0.03 compared with indicated group. (D) Beas2B cells were transfected with control vector and LC3B overexpression clones. After 24 to 30 hours, cells were exposed to hyperoxia. After 48 hours, cell viability was determined. Results are expressed as means ± SEM of three independent experiments. *P < 0.05.

Schemata of proposed pathways. Autophagy promotes cell survival after hyperoxia via interacting with Fas-mediated apoptosis pathways. Hyperoxia induces LC3B expression, a key initiator of autophagy. In this study, we describe dynamic interactions between LC3B and the apoptotic regulator Fas. Forced expression of LC3B was found to diminish apoptotic pathway initiation by the Fas-dependent death-inducing signaling complex. The interactions of LC3B and Fas are dependent on caveolin-1 and its phosphorylation state at Y-14. The balance between LC3B/cav-1/Fas complex determines the fate of the cell.