A20 inhibits osteoclastogenesis via TRAF6-dependent autophagy in human periodontal ligament cells under hypoxia

Abstract

Objectives: A20 exerts an anti-osteoclastogenic effect through the inhibition of NF-κB signalling in periodontitis. It also regulates autophagy via ubiquitin modification. This study was aimed at exploring the relationship between A20 and autophagy in anti-osteoclastogenesis in human periodontal ligament cells (hPDLCs) under hypoxia. Materials and Methods: Real-time PCR and Western blot were used to detect relative mRNA and protein levels. The formation of autophagosomes was measured by TEM. Osteoclastic differentiation was assessed by TRAP staining and hydroxyapatite resorption assay. The interactions between different proteins were observed by co-IP. Results: Cells cultured under 2% O₂ exhibited decreased A20 expression and increased RANKL/OPG (R/O) ratio. There was a negative correlation between A20 and TRAF6, and similar results were found with autophagic flux. A20 delayed the increase in R/O ratio under hypoxia. Autophagy in hPDLCs and osteoclast differentiation and hydroxyapatite resorption areas in mouse bone marrow mononuclear cells (BMMCs) were inhibited by A20. Moreover, inhibition of autophagy using 3-MA resulted in increased expression of A20 and decreased number and function of osteoclasts. In addition, A20 inhibited polyubiquitination at K63 and enhanced that at K48 in TRAF6 to suppress autophagy under hypoxic conditions. Conclusions: A20 inhibits osteoclastogenesis via inhibition of TRAF6-dependent autophagy in hPDLCs under hypoxia. These findings suggest that A20 may be a key gene target during bone loss in periodontitis via TRAF6-mediated inhibition of autophagy.