Objectives: Deubiquitinase Ubiquitin Specific Protease 1 (USP1) is essential for bone formation, but how USP1 regulates bone formation in response to oxidative stress remains unclear. In this study, we aim to investigate the biological function of USP1 in osteoblastic MC3T3-E1 cells.
Methods: Hydrogen peroxide (H2O2) as an oxidative reagent was used to trigger osteoblastic MC3T3-E1 cellular damage. Flow cytometry was used to evaluate ROS production, apoptosis, and pyroptosis. Real-time PCR and western bolt assay were used to detect the mRNA and protein levels of USP1. Moreover, coimmunoprecipitation was used to validate the relationship between USP1 and TRAF6.
Results: We demonstrated that USP1 was significantly decreased in MC3T3-E1 cells after H2O2 treatment. Overexpressing USP1 restored H2O2-decreased alkaline phosphatase activity and reactive oxygen species production. USP1 overexpression inhibited cytokine release and NLP3 inflammasome activation, which was mediated by NF-κB. Overexpressing USP1 prevented NF-κB translocation. USP1 formed a complex with TRAF6, inhibiting TRAF6 ubiquitination.
Conclusion: USP1 exhibits protective role in MC3T3-E1 cells by suppressing NF-κB-NLRP3 mediated pyroptosis in response to H2O2. The involvement of USP1 and TRAF6 in NLRP3 inflammasome signaling suggests a future therapeutic potential to improve clinical symptoms in osteoporosis.
Keywords: NF-κB; NLRP3; Pyroptosis; TRAF6; USP1.