Inhibition of LPL suppresses the osteoclast differentiation of bone-marrow-derived macrophages by promoting the ACSL4 ubiquitination

Background: The imbalance between osteoblast (OB) -led bone formation and osteoclast (OC) -induced bone resorption is a recognized reason of osteoporosis. However, further gene-related pathogenesis remains to be elucidated.

Methods: The microarray profile GSE225974 was used to identify the differentially expressed genes (DEGs) between OC and peripheral blood mononuclear cells (PBMC). Bone-marrow-derived macrophages (BMMs) treated with 30 ng/ml macrophage-colony-stimulating factor (M-CSF) and 100 ng/ml receptor activator of NF-kappa B ligand (RANKL) was to induce osteoclastic differentiation in vitro. The expression of lipoprotein Lipase (LPL) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting methods. Meanwhile, the regulatory role of LPL on osteoclastic differentiation was evaluated by monitoring Cathepsin K levels and TRAP staining. Proteins related to LPL were obtained by STRING, and the interaction between proteins was verified by immunoprecipitation (IP) and ubiquitination analysis.

Results: LPL was markedly up-expressed in OCs. Inhibition of LPL suppressed osteoclast differentiation of BMMs by inhibiting Cathepsin K and number of TRAP-positive cells. Then the results of STRING demonstrated that proteins related to LPL including the lipid synthesis gene ACSL4. Erastin treatment prominently weakened the effects of si-LPL on Cathepsin K levels and TRAP staining intensity by activating Ferroptosis. Mechanically, inhibition of LPL suppressed osteoclast differentiation by promoting ubiquitination levels of ACSL4, and over-expression of USP14 reversed the effects of LPL knockdown on regulating ubiquitination of ACSL4.

Conclusion: Suppression of LPL inhibits the osteoclast differentiation of BMMs in vitro. The mechanism may be related to the LPL knockdown induced USP14 meidated the ACSL4 ubiquitination. Taken together, down-regulation of LPL may be a promising method to suppress osteoclast differentiation to treat osteoporosis.

ACSL4; Ferroptosis; LPL; Osteoclastic differentiation; Osteoporosis; Ubiquitination.