USP14 promotes pyroptosis of human annulus fibrosus cells derived from patients with intervertebral disc degeneration through deubiquitination of NLRP3

Abstract

Intervertebral disc degeneration (IVDD) is a general disorder that results in low back pain and disability among many affected individuals. However, the current treatments for IVDD are limited to relieving the symptoms but do not solve the fundamental issue. In this study, the role of USP14 in mediating the activation of the NLRP3 inflammasome and the pyroptosis of AF cells from IVDD patients is determined in vitro, and gain- and loss-of-function assays of USP14 and the NLRP3 inflammasome are conducted. Pyroptosis of AF cells is detected by flow cytometry. The inflammatory cytokines (IL-1β and IL-18) and protein levels of NLRP3, active Caspase-1, Aggrecan, MMP3 and ADAMTS-5 are determined by ELISA and western blot analysis, respectively. The correlation between USP14 and NLRP3 is measured by coimmunoprecipitation and ubiquitination analysis. Upregulation of USP14 is accompanied by increased level of the NLRP3 inflammasome in AF cells from IVDD patients; furthermore, a positive correlation between them is observed. USP14 knockdown inhibits pyroptosis in AF cells by inducing ubiquitination of NLRP3, while overexpression of USP14 has the opposite effect, which is inhibited by the NLRP3 inflammasome inhibitor INF39. USP14 exerts its positive regulatory effect on AF cell pyroptosis by modulating the NLRP3/Caspase-1/IL-1β and IL-18 signaling axes.

Keywords

• Intervertebral disc degeneration
• USP14
• NLRP3 inflammasome
• deubiquitination
• pyroptosis