IL-32 Induces Epithelial-mesenchymal Transition in Human Pulmonary Alveolar Epithelial Cells by Triggering Endoplasmic Reticulum Stress


 BackgroundEpithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF.MethodsHuman lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting.ResultsStimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA. ConclusionsThese results suggest that IL-32 induces EMT in A549 cells by triggering ER stress. These results suggest that IL-32 may be a novel marker for IPF.


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Idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by continuous scarring of the lungs, can lead to a progressive decline of lung function and respiratory failure, with consequently high mortality rates [1]. The incidence of IPF has shown an increasing trend in recent years. However, since the precise cause and pathogenic mechanism are still unknown, there is a lack of effective treatment measures for IPF. Consequently, the median survival of IPF patients after diagnosis is only 2-3 years, and the mortality rate is higher than that of many cancers [2]. Therefore, in-depth investigations of the molecular mechanisms of IPF are of great significance for guiding the development of novel strategies for the prevention and treatment of the disease.
The onset of IPF involves disruption of the apoptosis/proliferation balance of fibroblasts, along with the excessive synthesis and accumulation of extracellular matrix components such as collagen [3]. Myofibroblasts are the primary source of collagen production, which are mainly regulated by the epithelial-mesenchymal transition (EMT) [4], a phenomenon in which epithelial cells acquire the phenotypic and biological characteristics of mesenchymal cells in response to certain stimuli [5]. Detailed studies have demonstrated that epithelial cells that have undergone EMT possess a contractile function and the ability to synthesize collagen [4], which are the key conditions for the onset and development of fibrosis. Thus, EMT appears to play an important role in IPF pathogenesis.
(TM) could induce ER stress and consequently trigger EMT through activation of histone deacetylase with accompanying interleukin (IL)-32 overexpression [11].
IL-32 is a recently discovered cytokine that plays a pivotal role in innate and acquired immunity through the regulation of T cells [12]. Previous studies have indicated that IL-32 can participate in the onset and development of many types of tumors by influencing the EMT processes of tumor cells [13][14][15]. Accordingly, we hypothesized that IL-32 might play a role in the pathogenic mechanism of IPF by influencing EMT. To test this possibility, we evaluated the role of IL-32 in A549 lung adenocarcinoma cells and the mechanisms of action in relation to the influence on EMT and ER stress. The results of this study could provide a scientific basis for IL-32 as a novel target in future research and clinical treatments of IPF.

Cell culture and treatment
Human lung adenocarcinoma A549 cells are an alveolar epithelial cell line with biological characteristics of AEC II, and were therefore used as an in vitro model of IPF. The A549 cells (ATCC R CRM-CCL-185 TM ) were purchased from American Type Culture Collection and cultured in RPMI-1640 culture medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a constant-temperature, constant-humidity incubator maintained at 37°C and 5% CO 2 . Six-well plates were inoculated with 1 × 10 6 cells each and cultured under normal or starvation conditions for 24 h. Subsequently, the cells were treated with 0.5 mg/ml TM, 1.

RNA interference
Small interfering RNA (siRNA) targeting IL-32 was designed and provided by Shanghai Genechem Co., Ltd. (Shanghai, China). Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA) was used to transfect A549 cells with plasmids containing IL-32 siRNA. Forty-eight hours after transfection, the cells were observed under an inverted fluorescence microscope to determine the transfection rate. Subsequently, RNA extraction was performed to confirm the interference efficiency via reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

RT-qPCR
Total RNA was extracted from the A549 cells using TRIzol reagent (Takara, Japan) in accordance with the manufacturer's instructions. Subsequently, the total RNA was reverse-transcribed to cDNA with the reverse transcription kit (TaKaRa). Using the

Western blotting
Total protein was extracted from the A549 cells and quantified using the BCA protein assay kit (Solarbio, Beijing, China). A sample containing 20 μg of protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Solarbio), transferred to a polyvinylidene fluoride membrane, and incubated with rabbit anti-mouse N-cadherin, GRP78, and α-SMA primary antibodies (Proteintech, Wuhan, China), or β-actin antibody (Bioss, Beijing, China) as a loading control, at room temperature for 2 h.
Subsequently, the membrane was washed, incubated with the horseradish peroxidaseconjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature, and exposed using the ECL kit. The experiment was repeated three times.

Statistical analysis
SPSS 20.0 software was used for statistical analysis of the experimental data. Quantitative data are expressed as the mean ± standard deviation, n=3, and comparisons between two groups were performed using the t-test. A significance level of α = 0.05 was adopted, with P < 0.05 indicating a statistically significant difference.

Tunicamycin treatment enhanced the Expression of IL-32 in A549 cells
Based on our previous study showing that TM can induce EMT in A549 cells, the cells were treated with 0.5 μg/ml TM for 24h [11]. Observation of the cells under an inverted microscope showed that the TM treatment induced significant cell death in a portion of the cells, with a lower cell number compared with that of the control group. Although the TMtreated cells showed no significant morphological changes, enlargement of intercellular spaces was evident, resulting in a looser arrangement of cells (Fig. 1a). Moreover, RT-qPCR showed that the mRNA expression level of IL-32 was significantly increased after 24 h of TM stimulation (P<0.05, Fig. 1b).

rhIL-32 induced EMT in A549 cells
After treatment with 100 ng/ml rhIL-32 for 24 h, a portion of the A549 cells showed a morphological change from a pebble-like shape to an irregular elongated shape (indicated by the red arrow in Fig. 2a). To ascertain if this phenomenon was caused by EMT, RT-qPCR was adopted to determine the influence of rhIL-32 treatment on the expression of EMTrelated molecules. Indeed, expression of the epithelial cell marker E-cadherin was significantly downregulated (P<0.05, Fig. 2b), while the expression of the mesenchymal cell markers N-cadherin, vimentin, and Zeb-1 were significantly upregulated (P<0.05, Fig.   2b) after rhIL-32 treatment.

rhIL-32 induced ER stress in A549 cells
Twenty-four hours after treatment of A549 cells with 100 ng/ml rhIL-32, both the mRNA and protein expression levels of GRP78 increased significantly (P<0.05, Fig. 4a, b, c), indicating that rhIL-32 can induce ER stress in A549 cells.
Moreover, treatment of rhIL-32 with 1.0 mM 4PBA significantly decreased the mRNA and protein expression levels of GRP78 compared with those of cells treated with rhIL-32 alone (P<0.05, Fig. 4a, b, and c).

ER stress mediated EMT in A549 cells
The aforementioned results suggest that rhIL-32 induces both EMT and ER stress in A549 cells. To further ascertain the relationship between ER stress and EMT, the expression of EMT-related molecules was measured in A549 cells treated with the ER stress inhibitor 4-PBA.
Compared with cells treated with rhIL-32 alone, 4-PBA treatment reduced the mRNA expression levels of the mesenchymal cell markers N-cadherin (P<0.05), snail (P<0.05), and Zeb-1 (P<0.05) (Fig 5a), and increased the mRNA expression level of the epithelial cell marker E-cadherin (P<0.05, Fig. 5a). Similarly, the protein expression of N-cadherin and α-SMA were upregulated in the rhIL-32 group but significantly downregulated in the rhIL-32 +4PBA group (P<0.05, Fig. 5b, c). Thus, EMT was inhibited following the inhibition of ER stress, indicating that rhIL-32 triggers ER stress and mediates the development of EMT in A549 cells.

IL-32 can induce inflammatory cytokine production in A549 cells
After rhIL-32 treatment, the mRNA expression levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, IL-1β, and IL-6( Fig. 5d) in the rhIL-32 group were significantly higher than those of the control group (P < 0.05). causes the onset of EMT in breast cancer cells [13]. Similarly, Lin et al. found that IL-32θ can inhibit EMT in colon cancer stem cells through the STAT3 signaling pathway [14].

Discussion
Other researchers have reported that IL-32α can inhibit the JAK2/STAT3 signaling pathway and reverse the IL-6-induced EMT process in pancreatic cancer cells [15]. The present study confirms and expands this role of IL-32, in demonstrating that it could induce EMT in pulmonary alveolar epithelial cells.
Some studies have found that ER stress induced by TM, thapsigargin, and mutations in SPC and SPA can mediate the development of EMT in pulmonary alveolar epithelial cells [9,10]. In addition, bleomycin could induce the onset of ER stress in a mouse model of pulmonary fibrosis, which in turn mediated the occurrence of EMT [23]. A similar conclusion was obtained in the present study, as IL-32 induced EMT through the induction of ER stress in pulmonary alveolar epithelial cells.
A multitude of factors have been suggested to trigger ER stress and consequently induce the development of EMT in pulmonary alveolar epithelial cells, including genetic and environmental factors such as smoking, chronic aspiration, viral infection, polymorphism in the promoter region of mucoprotein 5B, telomerase gene mutations, and mutations in SPC and SPA [19,24,25]. However, the exact mechanisms remain unclear. Although IL-32 appears to play a role in this process, we have not made clear which subtype of IL-32 plays a key role in it, in-depth investigation of these mechanisms and the role of each subtype of IL-32 will be the next step in our research.
The ER stress inhibitor 4-PBA was reported to induce EMT by TM-induced ER stress in renal tubular epithelial cells [26], and could also inhibit the expression of the EMT downstream markers GRP78 and LC3B-II in breast cancer cells [27]. Moreover, 4-PBA can alleviate atherosclerosis and stabilize existing plaques in mice through inhibition of ER stress [28].
Although similar effects of 4-PBA were observed in the present study by inhibiting TMinduced EMT, N-cadherin expression was not significantly reduced compared with the rhIL-32 treatment group. This could be attributed to the relatively short time of 4-PBA treatment in the experiment, which may have been insufficient for 4-PBA to exert its inhibiting effects on N-cadherin expression.
In addition, ER stress can promote the protein expression of LTBP1 and LTBP4, which are closely related to the secretion of TGF-β1 and TGF-β4 in pulmonary epithelial cells, thereby playing a key role in the development of pulmonary fibrosis [33]. The present study showed that rhIL-32 can induce ER stress and consequently upregulate TGF-β1, TNFα, IL-6, and IL-1β expression. Further research will be conducted to determine if the overexpression of these cytokines influences upstream pathways.

Conclusion
This study found that IL-32 can induce EMT in pulmonary alveolar epithelial cells by triggering ER stress offers a new approach for studies on IPF. In future research efforts, we will conduct an in-depth investigation of the relevant mechanisms in an attempt to establish IL-32 as a novel target for the treatment of pulmonary fibrosis.