Enhancement of the acrolein-induced production of reactive oxygen species and lung injury by GADD34

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by lung destruction and inflammation. As a major compound of cigarette smoke, acrolein plays a critical role in the induction of respiratory diseases. GADD34 is known as a growth arrest and DNA damage-related gene, which can be overexpressed in adverse environmental conditions. Here we investigated the effects of GADD34 on acrolein-induced lung injury. The intranasal exposure of acrolein induced the expression of GADD34, developing the pulmonary damage with inflammation and increase of reactive oxygen species (ROS). Conversely, the integrality of pulmonary structure was preserved and the generation of ROS was reduced in GADD34-knockout mice. Acrolein-induced phosphorylation of eIF2α in GADD34-knockout epithelial cells by shRNA protected cell death by reducing misfolded protein-caused oxidative stress. These data indicate that GADD34 participates in the development of acrolein-induced lung injury.

Figure 1

NAC prevents acrolein-induced lung injury and inflammation. Wild-type mice were intranasally instilled by 5 μmol/kg acrolein with intravenous injection of 100 μL of NAC (500 mg/kg) or equal volume of PBS. (a) Level of ROS in lung tissues was measured by DCFH-DA. (b) H&E staining of lung tissues (scale bar: 50 μm). (c) Alveolar macrophages were stained with PE-conjugated anti-CD11c and APC-conjugated anti-F4/80 and detected by FACS. (d) The expression of phospho-NF-κB p65 in the lung tissues by western blot.

Figure 2

Acrolein activates ER stress in lung tissue. Protein expression of ER stress was analyzed. Wild-type GADD34-knockout mice were intranasally instilled by 5 μmol/kg acrolein. Lung tissues were collected at the indicated times for western blot analysis.

Figure 3

GADD34 mediates acrolein-caused lung injury. The wild-type and GADD34-knockout mice were intranasally instilled by 1 and 5 μmol/kg acrolein. The mice were treated daily for 5 d/week for up to 28 days and then were sacrificed at 7 and 28 days. (a) The whole lungs of wild-type and GADD34-knockout mice were photographed at days 7 and 28. (b) H&E staining of lung tissue. Scale bar: 50 μm. Analysis of alveolar length determined by mean linear intercepts (n = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-type and GADD34-knockout mice (n = 4 mice in each group). (c) Lungs stained for epithelial type II cells (ProSpC green), nuclei (DAPI blue), and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields, n = 4). Scale bar: 50 μm. ^* P < 0.05, ^** P < 0.01. Data are represented as means ± s.e.m. (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice were measured by DCFH-DA after acrolein treated.

Figure 4

Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein. Lungs were collected from wild-type and GADD34-knockout mice at days 7 and 28 after 5 μmol/kg acrolein instillation. (a) Alveolar macrophages as F4/80^highCD11c⁺ and (b) neutrophils as Gr-1⁺CD11b⁺ were confirmed by FACS. (c) The expression of phospho-NF-κB p65. (d) The expressions of macrophage type I markers, TNFα, IL-6, and Irf5, and macrophage type II markers, Arg-1, Mrc-2, and Retnla, were analyzed by quantitative real-time PCR. (e) Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 μM acrolein for 12 and 24 h. Supernatants were taken and IL-6 expression was analyzed by ELISA. Data shown are the mean ratios ± SE of three separate experiments. Data are represented as means ± s.e.m. ^* P < 0.05, ^** P < 0.01.

Figure 5

GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell death. The 25 μM acrolein-treated shcon/LLCs and shGADD34/LLCs were analyzed. (a) Cell survival after 25 μM acrolein treatment was measured. ^* P < 0.05. (b) Levels of ROS production in shcon/LLCs and shGADD34/LLCs were measured by DCFH-DA after 25 μM acrolein treated for 8 h. (c) Cells were collected at the indicated times and protein expressions of ER stress signaling were detected by western blot. (d) The 25 μM acrolein-treated shcon/LLCs and shGADD34/LLCs with 20 mM NAC or without NAC for 8 h. Cells were stained with Annexin V-PE/7-amino-actinomycin D (7AAD). (e) Bands of proteins were analyzed by SDS-PAGE after 25 μM acrolein treatment. (f) Protein concentration was measured by Bio-Rad Protein Assay. (g) The shcon/LLCs and shGADD34/LLCs were treated by 25 μM acrolein with 10 μM MG132. Cells were collected and lysate was analyzed by SDS-PAGE. (h) Amount of proteins was measured by Bio-Rad Protein Assay. Data are represented as means ± s.e.m. ^* P < 0.05.

Figure 6

Protein synthesis promotes ROS production and cell death. The shcon/LLCs and shGADD34/LLCs were treated by 25 μM acrolein with 10 μM MG132 and/or 1 μg/mL CHX or without these agents for 12 h. (a) ROS levels were measured by DCFH-DA fluorescence by flow cytometry. Right, mean fluorescence after subtracting autofluorescence. ^* P < 0.05, ^** P < 0.01. (b) Cells were stained with Annexin V-PE/7-AAD and then analyzed by flow cytometry. ^* P < 0.05, ^** P < 0.01.