USP9X promotes LPS-induced fibroblast cell apoptosis, inflammation and oxidative stress by regulation of TBL1XR1 deubiquitination

Abstract

Abstract Background Ubiquitination and deubiquitination are involved in the progression of human diseases, including acute pneumonia. In this study, we aimed to explore the functions of ubiquitin-specific peptidase 9X-linked (USP9X) in lipopolysaccharide (LPS)-treated WI-38 cells. Methods WI-38 cells were treated with LPS to induce the cellular damage and inflammation. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay were performed to examine the proliferation of LPS-treated WI-38 cells. Flow cytometry analysis was conducted to detect LPS-treated WI-38 cell apoptosis. ELISA kits were utilized to determine the concentrations of inflammatory factors (IL-1β and TNF-α). Superoxide dismutase (SOD) activity and reactive oxygen species (ROS) level were examined with related kits. Ubibrowser (http://ubibrowser.bio-it.cn/ubibrowser/), ubiquitination assay and Co-Immunoprecipitation (Co-IP) assay demonstrated the interaction between USP9X and transducin β-like 1X related protein 1 (TBL1XR1). qRT-PCR assay and western blot assay were manipulated to determine the expression of USP9X and TBL1XR1. TBL1XR1 and USP9X knockdown experiments were conducted to explore their functions on LPS-induced WI-38 cell injury and inflammation. Results TBL1XR1 expression was upregulated in LPS-treated WI-38 cells. TBL1XR1 knockdown promoted cell proliferation and repressed apoptosis, inflammation and oxidative stress in LPS-treated WI-38 cells. Moreover, USP9X deubiquitinated TBL1XR1 to regulate TBL1XR1 expression. USP9X knockdown restored the effects of LPS on WI-38 cell proliferation, apoptosis, inflammation and oxidative stress, but these effects of USP9X knockdown were further abolished by TBL1XR1 overexpression. Additionally, USP9X promoted the NF-κB signaling pathway by the deubiquitination of TBL1XR1. Conclusion USP9X promoted the apoptosis, inflammation and oxidative stress of LPS-stimulated WI-38 cells through the deubiquitination of TBL1XR1.