Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

Yueguo Wang,Xu Wu,Jilong Shen,Huimei Wu,Shuang Ji,Guanghe Fei,Li Zhang,Zhenxing Ding,Youhui Tu,Wenying Zhang

doi:10.1155/2022/6666022

Abstract

NADPH oxidase 4 (Nox4) is an important source of reactive oxygen species (ROS) production, and its expression is increased in lipopolysaccharide- (LPS-) stimulated lung epithelial cells. Polymerase δ-interacting protein 2 (Poldip2) has been proved to bind Nox4 and participates in oxidative stress and inflammation. However, the role of Poldip2/Nox4 in LPS-induced oxidative stress and inflammation in lung epithelial cells remains unclear. Cell viability was measured via MTT assays. The expression of Poldip2, Nox4, heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX-2), AKT, and p-AKT was detected by Western blotting and/or immunofluorescence. Poldip2 and Nox4 interaction was analyzed via coimmunoprecipitation (Co-IP) assay. NADPH enzymatic activity and production of ROS, prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were assessed simultaneously. The small interfering RNA (siRNA) or plasmid targeting Nox4 was used to downregulate or upregulate Nox4, and the lentiviral vector encoding Poldip2 was used to downregulate or upregulate Poldip2. The present study demonstrated that LPS stimulation significantly increased the protein levels of Poldip2 and Nox4 and proved that Poldip2 interacted with Nox4 proved by Co-IP. Importantly, Poldip2 acted as an upstream regulator of Nox4. The increased expression of Nox4 and COX-2; NADPH enzymatic activity; production of ROS, PGE2, TNF-α, and IL-1β; and decreased HO-1 expression were significantly suppressed by lentiviral Poldip2 shRNA downregulation but were increased by lentiviral upregulation of Poldip2. Furthermore, inhibiting of PI3K-AKT signaling notably attenuated LPS-induced Poldip2/Nox4 activation. Our study demonstrated that Poldip2 mediates LPS-induced oxidative stress and inflammation via interaction with Nox4 and was regulated by the PI3K-AKT signaling. Targeting Poldip2 could be a beneficial therapeutic strategy for the treatment of ALI.

Highlights

Lipopolysaccharide (LPS), a major constituent of the cell walls of Gram-negative bacteria, plays a vital role in the development and progression of acute lung injury (ALI) [1]
Our data demonstrated that LPS induced oxidative stress and inflammation in lung epithelial cells by increasing the expression of NADPH oxidase 4 (Nox4) and COX-2 and NADPH
Our findings reveal that Poldip2 mediates oxidative stress and inflammation via interaction with Nox4 and such effect of Poldip2/Nox4 is regulated by the PI3K-AKT signaling in LPS-stimulated lung epithelial cells

Summary

Introduction

Lipopolysaccharide (LPS), a major constituent of the cell walls of Gram-negative bacteria, plays a vital role in the development and progression of acute lung injury (ALI) [1] It can initiate production of biologically active molecules from a variety of cells [2, 3], including inflammatory cytokines and reactive oxygen species (ROS). Studies showed that LPS markedly increased NADPH oxidase activation, including Nox, and intracellular ROS production in human lung epithelial cells [10] and the mouse preclinical model of ALI [11]. These observations indicate that Nox4-mediated oxidative stress and inflammation may be enhanced by LPS stimulation and implicated in LPSinduced ALI. The mechanism of the activation of Nox and Nox4-mediating effects during LPS stimulation is not completely understood

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