Particulate matter 2.5 induces autophagy via inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin kinase signaling pathway in human bronchial epithelial cells

Abstract

Particulate matter 2.5 (PM2.5) is a significant risk factor for asthma. A recent study revealed that autophagy was associated with asthma pathogenesis. However, the specific mechanisms underlying PM2.5-induced autophagy in asthma have remained elusive. In the present study, PM2.5-induced autophagy was evaluated in Beas-2B human bronchial epithelial cells and the potential molecular mechanisms were investigated. Using electron microscopy, immunofluorescence staining and immunoblot studies, it was confirmed that PM2.5 induced autophagy in Beas-2B cells as a result of PM2.5-mediated inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway in Beas-2B cells. LY294002, a PI3K inhibitor, reduced the accumulation of microtubule-associated protein 1 light chain 3 II and attenuated the effect of PM2.5. Phosphorylated (p-)p38, p-extracellular signal-regulated kinase and p-c-Jun N-terminal kinase were dephosphorylated following exposure to PM2.5. The roles of p53, reactive oxygen species scavenger tetramethylthiourea and autophagy inhibitor 3-methyladenine in PM2.5-induced autophagy in Beas-2B cells were also investigated. The results suggested that the PI3K/Akt/mTOR signaling pathway may be a key contributor to PM2.5-induced autophagy in Beas-2B cells. The results of the present study therefore provided an a insight into potential future clinical applications targeting these signaling pathways, for the prevention and/or treatment of PM2.5-induced lung diseases.

Figure 1

Autophagy is induced in Beas-2B cells following exposure to PM_2.5. (A) Beas-2B human bronchial epithelial cells were treated with the indicated concentrations of PM_2.5 for 24 h or with 100 µg/ml PM_2.5 for the indicated time-periods. Cell lysates were subjected to immunoblot analysis for detection of LC3 levels and β-actin was used as loading control. Quantification of the results are presented as the amount of LC3II normalized against LC3I. (B) Beas-2B cells were transfected with mCherry-eGFP-LC3 and treated with 100 µg/ml PM_2.5 for 24 h. Beas-2B cells were treated with phosphate-buffered saline and rapamycin as negative and positive controls, respectively. Cells were examined by fluorescent microscopy, and representative cells were selected and photographed. (C) PM_2.5 induced ultrastructural features of autophagy. Beas-2B cells were treated with 100 µg/ml PM_2.5 for 24 h and processed for electron microscopy. Note the double membrane structure of the autophagic vacuoles. Degrading autophagic vacuoles (AVds) are indicated. Scale bar, 500 nm. Values are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control. All above experiments were repeated three times. PM_2.5, particle matter 2.5; LC3, microtubule-associated protein 1 light chain 3; NC, normal control; N, nucleus; Mit, mitochondria; eGFP, enhanced green fluorescent protein; AV, autophagic vesicles.

Figure 2

The AMPK signaling pathway positively regulates PM_2.5-mediated autophagy in Beas-2B cells. (A) Western blot analysis of AMPK, p-AMPK and LC3II expression. (B) Quantification relative to β-actin expression. Values are expressed as the mean ± standard deviation of ≥3 separate experiments. Cells were treated with 100 µg/ml PM_2.5 for 24 h, following 2 h pre-treatment with the AMPK inhibitor DM (40 µm/l). Dimethyl sulfoxide was tested as a control. PM_2.5 enhanced AMPK phosphorylation in Beas-2B cells; however, blocking AMPK activation did not significantly influence PM_2.5-mediated autophagy. ^*P<0.05, vs. control. DM, dorsomorphin; PM_2.5, particle matter 2.5; AMPK, adenosine monophosphate-activated protein kinase; p-, phosphorylated; LC3II, microtubule-associated protein 1 light chain 3 II.

Figure 3

Role of the PI3K/AKT/mTOR signaling pathways in PM_2.5-induced autophagy. Cells were treated with PM_2.5 (0, 12, 24 or 48 h) and the expression levels of the indicated proteins were analyzed by immunoblotting. (A) Phosphorylation status of AKT and protein expression levels of AKT were examined by western blot assay in PM_2.5-treated Beas-2B cells. (B) Cells were treated with 100 µg/ml PM_2.5 for 24 h following 2 h pretreatment with the inhibitor, LY294002. The phosphorylation status of AKT and protein expression level of AKT were analyzed by western blotting in PM_2.5-treated Beas-2B cells. (C) Cells were treated with 100 µg/ml PM_2.5 for 24 h, following 2 h pre-treatment with rapamycin. The phosphorylation status of mTOR and protein expression levels of mTOR were analyzed by western blotting in PM_2.5-treated Beas-2B cells. Values are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control. PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; PM_2.5, particle matter 2.5; p-, phosphorylated; RAPA, rapamycin; LC3II, microtubule-associated protein 1 light chain 3 II.

Figure 4

Role of MAPK signaling pathways in PM_2.5-induced autophagy (A and C) Western blot assays were used to examine the total and phosphorylated protein levels of ERK, JNK and β-actin. (B) Western blot assays were used to examine the expression of LC3II/LC3I, ERK and p-ERK when ERK inhibitor U0126 (5 µM) was added for 1 h before PM_2.5 treatment. (D) Western blot assays were used to examine the expression of LC3II/LC3I, p38 and p- p38 when p38 inhibitor SB203580 (10 µM) was added for 1 h before PM_2.5 treatment. (E) Western blot assays were used to examine the expression of LC3II/LC3I when JNK inhibitor SP600125 (50 µM) was added for 1 h before PM_2.5 treatment. Values are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control. MAPK, mitogen activated protein kinase; PM_2.5, particle matter 2.5; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LC3, microtubule-associated protein 1 light chain 3; p-, phosphorylated.

Figure 5

PM_2.5-mediated autophagy is p53-independant. (A) Beas-2B cells were treated with PM_2.5 in the absence or presence of PFT-α for 24 h. LC3 and p-p53/p53 protein expression levels were evaluated by western blot analysis (n=3). β-actin was used as an internal control. (B) Beas-2B cells were treated with PM_2.5 in the absence or presence of TMTU for 24 h. LC3 protein expression levels were determined by western blot analysis (n=3). β-actin was used as an internal control. (C) Propidium iodide staining for PM_2.5-induced apoptosis was measured using a flow cytometer, with or without pretreatment of 3-MA (10 mM) and PM_2.5 (100 µg/ml). Values are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^***P<0.001 vs. control. PM2.5, particle matter 2.5; PFT-α, pifithrin-α; LC3, microtubule-associated protein 1 light chain 3; TMTU, tetramethylthiourea; 3-MA, 3-methyladenine.