In vitro study of the role of FOXO transcription factors in regulating cigarette smoke extract-induced autophagy.

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.