Anisomycin

Silencing of Gal-7 inhibits TGF-β1-induced apoptosis of human airway epithelial cells through JNK signaling pathway

Abstract
Apoptosis of epithelial cells is regarded as the initial pathological process of many lung diseases, including asthma. Previous studies have identified that galectin-7 (Gal-7), a regulator of apoptosis, was overexpressed in bronchial epithelial cells in asthma. However, the effect and mechanism of Gal-7 in the progression of asthma is still unclear. In this study, we investigated the expression and role of Gal-7 in the apoptosis of bronchial epithelial cells BEAS-2B upon TGF-β1 stimulation. TGF-β1 significantly induced apoptosis of BEAS-2B cells, as determined by flow cytometry. Western blot results revealed that the mRNA and protein expression of Gal-7 were obviously increased after TGF-β1 stimulation. Small interfering RNA (siRNA)-mediated knockdown of Gal-7 abrogated TGF-β1-evoked cell apoptosis. Simultaneously, increased Bcl-2 expression, decreased Bax expression and the cleavage of poly ADP-ribose polymerase (PARP) and caspase-3 activity were also monitored in TGF-β1-treated cells after Gal-7 siRNA transfection. Gal-7 silence also inhibited TGF-β1-induced c-Jun N-terminal kinase (JNK) phosphorylation in BEAS-2B cells. Furthermore, anisomycin, a specific activator for JNK, reversed the effect of Gal-7 siRNA on cell apoptosis induced by TGF-β1. These results demonstrate that Gal-7 silence attenuates TGF-β1-induced apoptosis in bronchial epithelial cells through the inactivation of JNK pathway. Therefore, Gal-7 may act as a potential target for asthma treatment.

Introduction
Allergic asthma is one of the most common chronic respiratory diseases that seriously affects the growth and health of children [1]. Although the pathogenesis of asthma currently remains unclear , there is increasing evidence showing that bronchial epithelium, a key element of the respiratory airways, plays a major role in the development of asthma [2]. The damage and shedding of airway epithelium is an important pathological feature of asthma. During the process of airway damage, the apoptosis and shedding of airway epithelial cells are inevitable. Chagani et al. had reported extensive epithelial cell apoptosis in severe asthma [3]. Therefore, abnormal apoptosis of airway epithelial cells plays an important role in the development of asthma. Transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, is produced by many cells and involved in a variety of cellular processes, including apoptosis [4, 5]. TGF-β1 has been reported to induce tissue injury [6] and apoptosis of cells, such as alveolar epithelial cells [7], gastric cancer cells [8] and human bronchial epithelial cells [4]. Increased TGF-β1 level was observed in bronchoalveolar lavage fluid of chronic asthmatic mice with peribronchial fibrosis [9] and also in asthmatic patients [10].

Several investigations supported that TGF-β1 was mainly involved in airway remodeling [11, 12]. High level of TGF-β1 expression was observed in the lung tissue in asthmatic rats. These findings demonstrate the key role of TGF-β in asthma.
Galectin-7 (Gal-7), a member of the galectins family that has affinity for β-galactosides, is encoded by gene LGALS7 located on chromosome 19q13.2 [13]. Gal-7 is differentially expressed by various tissues and all types of stratified epithelia [14], and has extensive biological activities [15]. Gal-7, a p53-induced gene, exerts a pro-apoptotic effect [16]. Gal-7 expression was increased after ultraviolet B irradiation, and was associated with the apoptotic process in ultraviolet radiation B-induced sunburn keratinocytes [16]. Recent research confirmed the high expression of Gal-7 in bronchial epithelial cells in asthma [17]. However, the relationship between Gal-7 and bronchial epithelial cell apoptosis is still elusive.Therefore, our study aims to investigate the effect of Gal-7 on the apoptosis of human bronchial epithelial cells induced by TGF-β1 stimulation and the underlying mechanism, providing a potential therapeutic target for asthma treatment.

1.Cell Culture and Treatments
Human bronchial epithelial cell line BEAS-2B was purchased from American Tissue Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37 ˚C in a humidified 5% CO2 atmosphere. When they reached 90% confluence, the cells were harvested and stimulated with different concentrations of TGF-β1 (0, 1, 5, 10 ng/mL) (Sigma, St. Louis, MO, USA) for 24 h.

2.Cell Transfection
Cells were transfected with human Gal-7-1 siRNA or Gal-7-2 siRNA (Thermo Fisher Scientific, Waltham, MA, USA) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The Gal-7 mRNA and protein level were respectively determined by qPCR and Western blot after transfection for 48 h.

3.Cell Counting Kit-8 (CCK-8) Assay
CCK-8 (Beyotime Technology, Jiangsu, China) was used to assess cell viability [18]. Briefly, cells were plated in 96-well plates (2×103 cells/well) with TGF-β1 (10 ng/mL) for 48 h. Subsequently, CCK-8 (10 μL) was added to each well. After 3 hours’ incubation, the absorbance at 450 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA).

4.Flow Cytometry Assay
Flow cytometry with Annexin V-FITC and propidium iodide (PI) double staining was performed to detect cell apoptosis [19]. Briefly, after washing three times with PBS, cells were suspended in 500 μL of 1× binding buffer, and incubated with 5 μL of FITC conjugated Annexin-V (Invitrogen) and 5 μL of PI (Invitrogen). After incubation in the dark for 15 min, the samples were analyzed by flow cytometry using a FacsCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

5.Caspase-3 Activity Assay
The caspase-3 activity was measured using a caspase-3 colorimetric assay kit (Abcam, Cambridge, MA) according to the manufacturer’s protocol [20].

6.Quantitative Real-time PCR (qPCR)
For qPCR analysis, total RNA of cells was extracted from cells by TRIzol reagent (Invitrogen). From each sample, 5 μg of RNA was reverse transcribed to cDNA using a miScript Reverse Transcription Kit (Qiagen, Dusseldorf, Germany). qPCR was carried out with SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA). The reaction was performed according to the following conditions: 3 min 95 ºC, then 35 cycles of 1 min 95 ºC, 1 min 60 ºC, 1 min 70 ºC, and 10 min 70 ºC extension. The primer sequences were as follows: Gal-7, forward: 5’ ACCAACCCGGTCCCAG-3’ and reverse: 5’-GCGGGCTAACGCTTTATTTGC-3’; internal control GAPDH, forward: 5’-AACGTGTCAGTOGTGGACCTG-3’ and reverse: 5’-AGTGGGTGTCGCTGTFGAAGT-3’. Relative gene expression was analyzed by the 2−ΔΔCt method.

7.Western Blot Analysis
Proteins were extracted using RIPA lysis buffer (Beyotime, China), and the protein concentrations were detected using the Bicinchoninic Acid (BCA) method. Equal amounts of protein were separated using 8% SDS-PAGE gels (Invitrogen). Subsequently, the protein was electrophoretically transferred to a PVDF membrane (Millipore, Schwalbach, Germany). Then the membrane was incubated for 1 h in 5% fat-free milk, and incubated with primary antibodies (goat antibody Gal-7 (1:1000; R&D Systems, Minneapolis, MN, USA); rabbit antibodies including B-cell lymphoma-2 (Bcl-2; 1:1000) and Bcl-2-associated X protein (Bax; 1:1000) (Santa Cruz Biotechnology, Waltham, MA, USA); poly ADP-ribose polymerase (PARP; 1:1000); phosphorylated JNK (p-JNK; 1:1000); JNK (1:1000) (Cell Signaling, Beverly, MA, USA) ) overnight at 4 ºC, and then incubated with secondary antibodies conjugated with horseradish peroxidase (1:3000; Santa Cruz Biotechnology) for an hour at room temperature. The bands were detected using enhanced chemiluminescence (ECL; Amersham, Little Chalfont, UK) after washing with PBST, and quantified by the Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).

8.Statistical Analysis
All the data are showed as mean ± standard deviation. SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze the differences between groups and among multiple groups by one-way ANOVA and Student’s t-test. P<0.05 was considered statistically significant difference. Results 1.Gal-7 was upregulated in human bronchial epithelial cells after TGF-β1 treatment TGF-β1-stimulated BEAS-2B cells showed a significant reduction in cell viability compared to untreated controls (P<0.05) (Fig. 1A). After incubation with 5 ng/mL and 10 ng/mL of TGF-β1 for 24 h, cell apoptosis was increased in BEAS-2B cells (Fig. 1B), suggesting that TGF-β1 induced apoptosis of human bronchial epithelial cells.To investigate the correlation between the Gal-7 and TGF-β1-triggered apoptosis of human bronchial epithelial cells, the expression of Gal-7 in TGF-β1-treated BEAS-2B cells was monitored. The mRNA (Fig. 1C) and protein (Fig. 1D) levels of Gal-7 was significantly up-regulated in TGF-β1-treated BEAS-2B cells compared with normal BEAS-2B cells, implying that Gal-7 may have a great influence on the apoptosis of bronchial epithelial cells. TGF-β1 (10 ng/ml) produced a significant decrease in cell viability but increased apoptosis and Gal-7 expression (P<0.01), therefore it was used for the subsequent experiments. 2.Gal-7 inhibition attenuated TGF-β1-induced apoptosis To explore the effect of Gal-7 on regulating the apoptosis of bronchial epithelial cells, BEAS-2B cells transfected with Gal-7-1 siRNA and Gal-7-2 siRNA. Compared with control group, Gal-7 mRNA and protein was decreased by 0.41-fold and 0.32-fold, respectively, in Gal-7-1 siRNA transfected group and they were decreased by 0.32-fold and 0.24-fold, respectively, in Gal-7-2 siRNA transfected group (Fig. 2A and Fig. 2B). As Gal-7-2 siRNA led to a greater reduction in Gal-7 than Gal-7-1 siRNA, it has been used in subsequent studies. After transfection of BEAS-2B cells with Gal-7 siRNA for 48 h and stimulated with TGF-β1 for 24 h, cell apoptosis was detected. Gal-7 silence obviously enhanced the cell viability of TGF-β1-treated BEAS-2B cells (Fig. 2C). As shown in Fig.2D, comparing to that of TGF-β1 treated BEAS-2B cells, Gal-7 siRNA remarkably inhibited TGF-β1-induced cell apoptosis. Gal-7 silence restrained the increase in Bax and the decrease in Bcl-2 in response to TGF-β1 (Fig. 2E). Gal-7 silence also significantly attenuated TGF-β1-induced PARP cleavage in BEAS-2B cells (Fig. 2E). In addition, Gal-7 silence inhibited the caspase-3 activity in TGF-β1-treated BEAS-2B cells (Fig. 2F). 3.Gal-7 silence suppressed TGF-β1-induced jnk signaling activation It was reported that the abnormal activation of JNK signaling pathway often caused cell apoptosis [21]. To determine the mechanism of Gal-7 on human bronchial epithelial cell apoptosis, we investigated the protein expression of JNK in BEAS-2B cells. TGF-β1 obviously induced JNK phosphorylation, whereas Gal-7 silence inhibited JNK activation that induced by TGF-β1 (Fig. 3). These data suggested that Gal-7 activated the JNK signaling pathway in BEAS-2B cells. 4.Gal-7 silence inhibited TGF-β1-induced apoptosis in BEAS-2B cells via blocking the jnk pathway To explore the effect of JNK pathway on Gal-7 cessation-mediated anti-apoptosis effects, BEAS-2B cells were treated with anisomycin (20 μg/mL /mL), a specific JNK activator, for 1 h [22]. Cells were divided into control group, TGF-β1 group (cells were incubated with 10 ng/mL TGF-β1 for 24 h), Gal-7-2 siRNA+TGF-β1 group (after transfected with Gal-7-2 siRNA for 48 h, cells were incubated with 10 ng/mL TGF-β1 for 24 h), TGF-β1+anisomycin group (after treated with 20 μg/mL anisomycin for 1 h, cells were incubated with 10 ng/mL TGF-β1 for 24 h), TGF-β1+Gal-7-2 siRNA+anisomycin group (cells were treated with 20 μg/mL anisomycin for 1 h and then exposed to 10 ng/mL TGF-β1 for 24 h after Gal-7-2 siRNA transfection). The cell apoptosis in TGF-β1+Gal-7-2 siRNA+anisomycin group was higher than that in TGF-β1+Gal-7-2 siRNA group, as shown in Fig. 4A. These indicate that anisomycin reversed the repression of Gal-7 siRNA on TGF-β1 triggered cell apoptosis. Moreover, The upregulation of Bax and cleaved PARP induced by TGF-β1 was downregulated by Gal-7 siRNA. And Gal-7 siRNA significantly upregulated the decrease of Bcl-2 induced by TGF-β1. Anisomycin abolished the effect of Gal-7 siRNA on the expression of Bax, Bcl-2 and cleaved PARP in response to TGF-β1 (Fig. 4B). Anisomycin also counteracted the inhibition of caspase-3 activity by Gal-7 siRNA in the presence of TGF-β1 (Fig. 4C). These results revealed that Gal-7 silence inhibited apoptosis through JNK signal inactivation in BEAS-2B cells in response to TGF-β1. Discussion Apoptosis, a type of programmed cell death, plays a critical role in pulmonary disease, such as asthma [23]. Increased apoptosis of bronchial epithelial cells was observed in childhood asthma [23]. TGF-β1 is a growth factor that promotes multiple cell apoptosis. TGF-β1 is elevated in asthmatic patients [24] and has been demonstrated to be participated in the development of airway remodeling in asthma [25]. The results above suggest that TGF-β1 may play a key role in asthma through increasing the rates of apoptosis of airway epithelial cells. It has been demonstrated that TGF-β1 induced apoptosis in the human bronchial epithelial cells, which will aggravate the development of asthma [4]. In this study, we used TGF-β1 to stimulate BEAS-2B cells. Consistent with the literature [4], this research also corroborated that TGF-β1-induced the apoptosis of human airway epithelial cells.Galectins are a family of β-galactoside-binding lectins and several galectins have been shown to be able to regulate cellular functions. Previous study has revealed that Gal-7 overexpression displayed pro-apoptotic function in HeLa and DLD-1 cells [14]. Gal-7 was also reported to be up-regulated in bronchial epithelial cells in asthma [17]. It was speculated that Gal-7 may be associated with bronchial epithelial cell apoptosis in asthma. Together with previous results, this study showed that Gal-7 was associated with cell apoptosis [16]. This study demonstrated that Gal-7 silence inhibited TGF-β1-induced apoptosis in airway epithelial cells. The effect of Gal-7 inhibition on TGF-β1-induced apoptosis was further verified by measuring the activity of caspase-3 and the expression of Bax, Bcl-2 and PARP. Apoptosis is regulated by the activation of effector caspases. Caspase-3 and its downstream substrate PARP play an important role in the activation of early apoptotic events. PARP cleavage has been considered as a crucial marker for the activation of functional caspase [26]. Furthermore, the PARP cleavage is an indicator of early apoptosis in asthmatic bronchial epithelium [27]. Research has revealed that Gal-7 is a new mitochondrial Bcl-2 interacting partner and may inactivate Bcl-2 by binding to it [28]. Transfection of Gal-7 siRNA also reduced caspase-3 activity and PARP cleavage, as well as Bax expression, and increased Bcl-2 expression. We subsequently elucidated the underlying molecular mechanism involved in Gal-7 inhibition on TGF-β1-induced apoptosis. Studies have shown that TGF-β can affect JNK signal pathway [29]. JNK is a stress-activated protein kinase and a member of the mitogen-activated protein kinase family [30]. It exerts pro-apoptotic effects during an apoptotic process [31, 32]. The JNK pathway was reported to play a significant role in the course of asthma airway remodeling [33]. Studies revealed that the protein expression of Wnt5a/JNK signaling pathway-related molecules was elevated in asthmatic rats [34]. The stimulation of JNK by TGF-β1 leads to phosphorylation of its substrate Jun at serine residues 63 and 73 [35]. Our data showed that JNK was activated after TGF-β1 treatment in the airway epithelial cells, and Gal-7 silence suppressed JNK activation. These results were consistent with the previous study, which demonstrated that Gal-7 activated JNK pathway to exhibit a pro-apoptotic effect on HeLa and DLD-1 cells. A further study indicated that the anti-apoptotic effects of Gal-7 siRNA in response to TGF-β1 were partially reversed when anisomycin, a specific JNK activator, activated JNK signaling. Therefore, our study suggests that Gal-7 plays a potential role in the regulation of TGF-β1-induced cell apoptosis through JNK signaling. However, it has not affirmed the effects of Gal-7 in vivo asthma model in our study. Further investigation in vivo will be performed. Conclusion In summary, this study demonstrated the essential role of Gal-7 in TGF-β1-induced cell apoptosis. Gal-7 deficiency inhibited cell apoptosis, caspase-3 activity, Bax and cleaved PARP expression, but up-regulated Bcl-2 expression. In addition, Gal-7 silence attenuated TGF-β1-induced apoptosis in BEAS-2B cells via blocking the JNK pathway. These results Anisomycin suggest that Gal-7 silence may ameliorate the damage of human bronchial epithelial cells by protecting against apoptosis. Gal-7 may be a potential target for the treatment of asthma.