SIRT7 inhibits osteoclast differentiation in osteoarthritis by mediating NDRG3 deacetylation to suppress the c-Raf/ERK signaling pathway

Abstract

Objective

Osteoarthritis (OA) is the most common type of arthritis, mainly triggered by inflammatory factors secreted by chondrocytes and subchondral osteoclasts. SIRT7, an NAD⁺ -dependent deacetylase, is downregulated in OA, but its role in osteoclast differentiation in OA and its underlying molecular regulatory mechanisms remain unclear.

Methods

Synovial fluid was collected from 22 patients with osteoarthritis (OA) and 16 healthy individuals. Primary murine bone marrow-derived macrophages (BMMs) were isolated and differentiated into osteoclasts using M-CSF and RANKL. Cell proliferation was assessed using CCK-8 assay. Osteoclast differentiation was evaluated by TRAP staining. Levels of osteoclast differentiation markers (NFATc1, CTSK, and c-FOS) were measured by qRT-PCR. Western blotting was performed to measure the protein level of SIRT7, NDRG3, c-Raf, p-c-Raf, p-ERK, and ERK. Immunoprecipitation (IP) was used to detect acetylation and ubiquitination levels on NDRG3, while co-immunoprecipitation (Co-IP) was employed to examine the interaction between SIRT7 and NDRG3.

Results

The expression level of SIRT7 in synovial fluid of OA patients is significantly reduced. Overexpression of SIRT7 markedly inhibits osteoclast differentiation. Additionally, NDRG3 expression is significantly increased in the synovial fluid of OA patients and negatively correlates with SIRT7 expression level. SIRT7 directly binds to NDRG3 protein, reducing its acetylation level and promoting its ubiquitination degradation, thereby inhibiting its expression. Knockdown of NDRG3 suppresses osteoclast differentiation, while overexpression of NDRG3 reverses the effect of SIRT7 on osteoclast differentiation. Furthermore, overexpression of SIRT7 suppresses the levels of p-c-Raf and p-ERK by targeting the downregulation of NDRG3 and thus inhibiting osteoclast differentiation.

Conclusion

SIRT7 downregulates NDRG3 expression via directly reducing its acetylation and promoting its ubiquitination degradation, which consequently inhibits the c-Raf/ERK signaling pathway and suppresses osteoclast differentiation in OA. This study provides new insights into the pathophysiology of OA, suggesting that SIRT7 and NDRG3 could be novel molecular markers for the diagnosis and treatment of OA.