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Lipopolysaccharide Induces Autophagic Cell Death through the PERK-Dependent Branch of the Unfolded Protein Response in Human Alveolar Epithelial A549 Cells

Background: Alveolar epithelial cell death plays a critical role in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury. Increased autophagy has a dual effect on cell survival. However, it is not known whether autophagy promotes death or survival in human alveolar epithelial cells exposed to LPS. Methods: Genetic and pharmacological approaches were used to evaluate the effect of autophagy on A549 cell viability upon LPS exposure. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways were examined with immunoblotting studies to further explore underlying mechanisms. Results: Treatment with LPS (50 µg/ml) led to autophagy activation and decreased cell viability in A549 cells. Blocking autophagy via short interfering RNA or inhibitor significantly decreased, whereas rapamycin increased, the LPS-induced effect on viability. ER stress was activated in LPS-stimulated A549 cells, and ER stress inhibitor reduced LPS-induced autophagy. LPS activated only the PERK pathway and had rarely effect on the ATF6 and IRE1 branches of the UPR in A549 cells. Moreover, the knockdown of PERK and ATF4 attenuated LPS-induced autophagy and promoted cell survival. Conclusion: In human alveolar epithelial A549 cells, LPS induces autophagic cell death that depends on the activation of the PERK branch of the UPR upon ER stress.


Introduction
Alveolar epithelial cell damage has been recognized as a prominent feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and is known to contribute to the pathogenesis of this syndrome [1,2]. The degree of alveolar epithelial injury has been considered to represent an important prognostic marker of ARDS [3]. Lipopolysaccharide (LPS), a component of the outer cell walls of Gram-negative bacteria, has been considered one of the important factors leading to ALI [4]. Previous studies suggested that uncontrolled primary contributor to diffuse alveolar damage [5]. However, therapeutic strategies aimed Accordingly, it is necessary to further characterise the functions and mechanisms through which LPS causes alveolar epithelial cell damage.
Autophagy is a fundamental cell biological process that is responsible for the turnover of long-lived proteins and cytoplasmic organelles through a lysosome-mediated degradation pathway. During autophagy, the cytosol and organelles such as mitochondria and the endoplasmic reticulum (ER) are engulfed into a double-or multi-membrane-bound vacuole (an autophagosome). The vacuole membrane subsequently fuses with a lysosome, in which the encapsulated components are degraded by lysosomal hydrolases, and the breakdown products are released for reutilization [9][10][11]. This self-digestion process has a dual effect on cell survival: cytoprotection and cytotoxicity. Evidence has been presented that autophagy and stimuli present, and the duration and strength of the stimulus exposure [12,13]. For instance, the autophagic protein microtubule-associated protein 1 light chain 3 (LC3B) can facilitate bronchial epithelial cell survival in the context of oxygen-dependent cytotoxicity dysregulation of the Akt-TSC2-mTOR signalling pathway in alveolar epithelial A549 cells autophagy exerts distinct effects on the survival of different cell types. Furthermore, for the same cell types, hypoxia-induced autophagy can promote survival if induced under conditions of well-controlled hypoxia and without metabolic stress but can induce cell death under conditions of severe hypoxia concomitant with nutrient investigated as a pathway of cell death in these studies. However, it is not well-known whether autophagy plays a role in either the initiation or inhibition of cell death processes when human alveolar epithelial cells are challenged with LPS.
Many sources of stress cause a perturbation in the ER, termed ER stress, which results in the accumulation of unfolded or misfolded proteins. This accumulation subsequently triggers the unfolded protein response (UPR), which enables the cell to either overcome the stress [19,20]. Under non-stress conditions, these sensors bind to the ER-resident glucoseand exist in their deactivated forms [21][22][23]. Upon stress, misfolded proteins in the ER lumina cause the UPR sensors to detach from the chaperones, then freeing sensors activate of ATF4 translation, leading to the transcription of ATF4's downstream targets, including has also been presented indicating that there is important crosstalk between the ER stress and autophagy pathways because autophagy can be used as a misfolded protein degradation triggered by the ER stress response to insults plays an important role in the activation of autophagy as well as in the determination of life-or-death cell fates [30]. However, whether LPS-induced autophagic activity is associated with ER stress and the UPR in A549 cells has not been established.
In the present study, using a cell-culture model of ALI [31][32][33], we provide novel evidence that LPS-induced autophagy promotes cell death in alveolar epithelial cells and

Materials and Methods
Cell culture and drug treatment The human type II lung epithelial A549 cell line was obtained from the American Type Culture heat-inactivated foetal bovine serum (Gibco) and incubated at 37 2 . LPS (0111:B4), 3-methyladenine or 3-MA (3 mM) or 4-PBA (2.5 mM) dissolved in PBS were used to pretreat the cells for 2 h before the administration of LPS dissolved in PBS. LPS was pre-incubated at 37 for 1 h with polymyxin B (100 g/ml) or PBS. The control cells received PBS or DMSO only.

Transfections and RNA interference
(constructed by GeneChem Co. Ltd, Shanghai, China) using Lipofectamine 3000 Transfection Reagent with LPS at 50 Leica Microsystems, Mannheim, Germany) co-transfected with EGFP-LC3 plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen) blotting analysis. In parallel, cells were treated with LPS and analysed as described above.
Transmission electron microscopy Cells were treated with LPS (50 temperature. The cells were then embedded, and ultra-thin sections were obtained. After staining with uranyl acetate/lead citrate, the sections were analysed by transmission electron microscopy (JEM-Cell death assay each well, and the plates were incubated for 2 h. Absorbance values at 450 nm were then recorded with a microplate reader. The proportion of cells per treatment group was normalized to the control wells.

Cellular Physiology and Biochemistry
Cellular Physiology and Biochemistry and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations.

Lactate dehydrogenase (LDH) assay
After treatment with LPS (50 of LDH leakage in the A549 cells was measured using a kit (Beyotime) according to the manufacturer's protocol.
Immunoblotting resulting fractions was measured using an enhanced BCA protein assay kit (Beyotime). The proteins were Perry Laboratories) were used to detect the primary antibodies. Bands were visualized with an enhanced chemiluminescence reagent (Perkin-Elmer Life Sciences)

Statistical methods
All values were expressed as the mean±SEM of the indicated number of experiments. The statistical P values of

LPS induced autophagy in A549 cells
To evaluate autophagy in LPS-treated A549 cells, proteins involved in the induction or progression of the autophagic pathway, including LC3B, Beclin-1 and Atg5, were detected by immunoblotting. Treating cells with LPS resulted in an increase in total LC3B expression and in the dose-and time-dependent accumulation of its active form, LC3B-II. Consistent with the observed increase in LC3B expression, Beclin-1 and Atg5 were upregulated after LPS substrate (Fig. 1A). To verify that the increased autophagy was due to the presence of LPS, LPS was pre-incubated with polymyxin B found that pre-incubation of LPS with polymyxin B decreased the formation of LC3B-II protein (Fig. 1B).
which is a marker of autophagosome formation. LPS treatment induced the formation of EGFPthese results revealed that the number of LC3 puncta per cross-sectioned cell increased after the LPS treatment relative to the control treatment (Fig. 1C, lower panel).
to identify morphological indices of autophagy via transmission electron microscopy. The with LPS, while this ultrastructural feature was rarely observed in the control cells (Fig. 1D).

LPS at 50 g/ml inhibited cell viability independent of apoptosis and necrosis in A549 cells
Treatment of the cells with LPS at concentrations of 50 ( Fig. 2A), 100, and 150 g/ml (data M) was used as ml LPS did not affect the rate of lactate dehydrogenase (LDH) leakage, a hallmark of cell that the LPS-induced decrease in cell viability is not attributable to apoptosis or necrosis. Therefore, we presume that other mechanism(s) may contribute to the observed cell death. LPS which was pre-incubated without or with polymyxin B *P< drogenase (LDH) leakage rate was measured by spectrophotometry as described in the Materials and methods section. The data are presented as the mean±SEM of three independent experiments. *P< P<0.01.  Beclin-1 or Atg5 (Fig. 3C). Furthermore, as shown in Fig. 3D and 3E, the knockdown of Beclin-1 or Atg5 decreased the expression level of LC3B-II protein and the number of LC3 puncta in the A549 cells treated with LPS. Moreover, the decrease in cell viability after exposure to LPS was Beclin-1 or Atg5 knockdown (Fig. 3F).

ER stress contributes to LPS-induced autophagy in A549 cells
To evaluate whether ER stress is responsible for the activation of autophagy in A549 and GRP94 proteins, in LPS-stimulated A549 cells. As illustrated in Fig. 4A, the protein , an ER lysed by immunoblotting with the indicated antibodies. The bar graph indicates the relative abundance of the LC3-II protein (normalized to that of GAPDH). *P < 0.05.

Cellular Physiology and Biochemistry
Cellular Physiology and Biochemistry stress inhibitor 4-PBA, which was accompanied by a decrease in the level of LC3B-II (Fig.  4B). The results indicate that ER stress contributed to the activation of autophagy in LPStreated A549 cells.
LPS activated the PERK-eIF2 -ATF4-GADD34 pathway of the UPR but had no effect on the ATF6 and IRE1-sXBP1 branches To assess whether all three branches of the UPR are activated upon LPS-induced stress in A549 cells, we analysed constituents involved in these three pathways of UPR signalling.

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The bar graph indicates the relative abundance of each protein (normalized to that of histone H1). (B) Total of each protein (normalized to that of GAPDH). *P < 0.05, **P < 0.01 compared to the control group.

Cellular Physiology and Biochemistry
The PERK-eIF -ATF4-GADD34 pathway is responsible for LPS-induced autophagy and cell death in A549 cells LPS-induced autophagy and cell survival in A549 cells. The transfection of the cells with an PERK or ATF4 PERK or ATF4

Discussion
Alveolar epithelial cell death is a prominent characteristic of the ALI induced by LPS current study, employing the cell culture model of ALI used by many research groups, which utilises A549 cells to examine the injury-defence mechanism of alveolar epithelial cells [31][32][33], we elucidated the effects of autophagy on the outcome of human alveolar epithelial cell fate in response to LPS and studied the underlying mechanisms. Autophagy, an evolutionarily conserved process, has primarily been characterized indicate that LPS can induce autophagy in A549 cells, as evidenced by the observation of both biochemical and morphological indices of autophagy in the LPS-treated A549 cells, including the enhanced expression of autophagic proteins, the decreased abundance of an autophagic substrate accompanied by the formation of double-membrane autophagosomes that were detected by electron microscopy, and the accumulation of EGFP-LC3 puncta in transfected cells observed with a laser confocal scanning microscope. The effect of LPS on autophagy in autophagic protein LC3B-II. Autophagy also has been considered to form part of a pro-death or a pro-survival pathway in a variety of models [42]. In some cases, autophagy represents an adaptive response that protects a cell from death induced by exogenous or endogenous stimuli [12, mechanism to promote a type of cell death that is termed autophagic cell death (also known which is often observed under extreme stress conditions, could be considered the outcome of failed adaptation [15]. Based upon our observations and these concepts, we hypothesized that autophagy may determine cell fate when human alveolar epithelial A549 cells are induced A549 cell death. In our model, LPS treatment induced autophagic cell death in the autophagy is an alternative or complementary mechanism by which LPS results in alveolar epithelial cell injury and contributes to the pathogenesis of ALI.
It should be noted that LPS was previously reported to induce apoptosis in A549 cells can be attributed to the varied duration and strength of the LPS exposure in the different experimental models used in these studies. Cells undergoing different degrees of stimulation induced by LPS may undergo different death patterns. Another possible explanation is that A549 cells were resistant to the induction of apoptosis in the absence of co-culturing the cells with polymorphonuclear leukocytes [31].
To date, the exact mechanism regulating the autophagy induced by LPS has not been important trigger of autophagy. Under most conditions, the UPR's three known sensors are coordinately activated [24]. In this study, we found that LPS initiated ER stress in A549 cells, to determine whether all three branches of the UPR were elicited following LPS-induced stress in A549 cells. Unexpectedly, we found that LPS treatment of A549 cells activated one According to recent evidence, various ER stressors result in the activation of distinct branches of the UPR accompanied by differential cellular effects in different cell types [30,51,52]. In addition, the impact of ER stress on cell fate depends on the level of activation of the pathway has been shown to play a neuroprotective role in a model of Parkinson's disease [53], whereas severe ER stress followed by a sustained and prolonged UPR that involves CHOP activation often leads to cell death [54]. Therefore, we further assessed whether the increased autophagy and decreased survival of LPS-treated A549 cells were associated with II and decreased the punctate accumulation of LC3, accompanied by a partial enhancement of the viability in the A549 cells exposed to a minimal cytotoxic dose of LPS. Our data indicate cell death when the A549 cells are challenged with a minimal cytotoxic dose of LPS. undergo autophagic cell death following a challenge with a minimal cytotoxic dose of LPS stress. These molecular events might represent another important mechanism contributing to the alveolar epithelial cell damage induced by LPS, which likely results in the pathogenesis for the management of alveolar epithelial cell death during ALI.