Bagam Prathyusha, Kaur Gagandeep, Singh Dhirendra Pratap, Batra Sanjay
Laboratory of Pulmonary Immunotoxicology, Environmental Toxicology Department, Health Research Center, College of Sciences and Engineering, Southern University and A&M College, Baton Rouge, LA, 70813, USA.
Cell Biol Toxicol. 2021 Aug;37(4):531-553. doi: 10.1007/s10565-020-09556-y. Epub 2020 Nov 2.
Cigarette smoking is the chief etiological factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by cigarette smoke (CS) causes protein degradation, DNA damage, and cell death, thereby resulting in acute lung injury (ALI). In this regard, autophagy plays a critical role in regulating inflammatory responses by maintaining protein and organelle homeostasis and cellular viability. Expression of autophagy-related proteins (ARPs) is regulated by the fork head box class O (FOXO) transcription factors. In the current study, we examined the role of FOXO family proteins-FOXO1 and FOXO3a-in regulating CS extract (CSE)-induced autophagy. Using human lung adenocarcinoma cells with type II alveolar epithelial characteristics (A549), we observed CSE-mediated downregulation of FOXO3a. In contrast, there was a pronounced increase in the expression of FOXO1 at both the transcriptional and translational levels in the CSE-challenged cells compared with controls. Interestingly, knockdown of FOXO3a heightened the CSE-mediated increase in expression of cytokines/chemokines (IL-6, IL-8, and MCP-1), ARPs, and the FOXO1 transcription factor. Moreover, FOXO1 knockdown rescued CSE-mediated upregulation of ARPs in A549 cells. In addition, using the ROS inhibitor N-acetyl-L-cysteine (NAC), we observed abrogated mRNA expression of several ARPs and production of inflammatory cytokines/chemokines (IL-6, IL-8, MCP-1, and CCL-5) in the CSE-challenged cells suggesting an important role of ROS in regulating CSE-induced autophagy. Chromatin immunoprecipitation of FOXO1 and FOXO3a demonstrated increased binding of the former to promoter regions of autophagy genes- BECLIN1, ATG5, ATG12, ATG16, and LC3 in CSE challenged cells. These findings suggest the role of FOXO1 in regulating the expression of these genes during CSE exposure. Overall, our findings provide evidence for FOXO3a-dependent FOXO1-mediated regulation of autophagy in the CSE-challenged cells. Graphical abstract.
吸烟是慢性阻塞性肺疾病（COPD）的主要病因。香烟烟雾（CS）诱导的氧化应激会导致蛋白质降解、DNA损伤和细胞死亡，进而引发急性肺损伤（ALI）。在这方面，自噬通过维持蛋白质和细胞器的稳态以及细胞活力，在调节炎症反应中起关键作用。自噬相关蛋白（ARP）的表达受叉头框O类（FOXO）转录因子调控。在本研究中，我们研究了FOXO家族蛋白——FOXO1和FOXO3a——在调节CS提取物（CSE）诱导的自噬中的作用。利用具有II型肺泡上皮细胞特征的人肺腺癌细胞（A549），我们观察到CSE介导的FOXO3a下调。相反，与对照组相比，在受到CSE刺激的细胞中，FOXO1在转录和翻译水平上的表达均显著增加。有趣的是，敲低FOXO3a会增强CSE介导的细胞因子/趋化因子（IL-6、IL-8和MCP-1）、ARP和FOXO1转录因子表达的增加。此外，敲低FOXO1可挽救CSE介导的A549细胞中ARP的上调。此外，使用活性氧（ROS）抑制剂N-乙酰-L-半胱氨酸（NAC），我们观察到在受到CSE刺激的细胞中，几种ARP的mRNA表达以及炎症细胞因子/趋化因子（IL-6、IL-8、MCP-1和CCL-5）的产生被消除，这表明ROS在调节CSE诱导的自噬中起重要作用。对FOXO1和FOXO3a进行染色质免疫沉淀显示，在受到CSE刺激的细胞中，前者与自噬基因——BECLIN1、ATG5、ATG12、ATG16和LC3的启动子区域的结合增加。这些发现表明FOXO1在CSE暴露期间调节这些基因表达中的作用。总体而言，我们的研究结果为CSE刺激的细胞中FOXO3a依赖性FOXO1介导的自噬调节提供了证据。图形摘要。
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