Inhibition of NEURL3 Suppresses Osteoclast Differentiation via BMP7 Ubiquitination Modulation

Osteoporosis (OP) is a genetic disorder characterized by an imbalance between osteoblast-mediated bone formation and osteoclast-induced bone resorption. However, the underlying gene-related mechanisms of its pathogenesis remain to be fully elucidated. Aberrantly expressed neuralized E3 ubiquitin-protein ligase 3 (NEURL3), which is related to osteoclastic differentiation, was identified through the analysis of the microarray profile GSE176265. Bone marrow-derived macrophages (BMMs) were isolated from the femurs and tibias of C57BL/6 J mice and treated with 30 ng/mL macrophage-colony-stimulating factor (M-CSF) and 100 ng/mL receptor activator of nuclear factor-kappa B ligand (RANKL) to induce osteoclastic differentiation, thereby mimicking OP in vitro. To model OP in vivo, ovariectomy (OVX)-induced bone loss was performed in mice. High expression levels of NEURL3 were confirmed in clinical samples, OP model cells, and OP model mice using quantitative real-time polymerase chain reaction (qRT-PCR). The impact of NEURL3 on osteoclastic differentiation was assessed by evaluating cell viability and the expression levels of osteoclastogenesis-related marker genes. Additionally, bone loss in mice was quantified using micro-computed tomography before and after NEURL3 inhibition. Mechanistically, the effects of NEURL3 on osteogenic differentiation were investigated by determining the protein levels of osteogenic markers via Western blotting. NEURL3 was markedly overexpressed in serum samples collected from patients with OP, OVX-induced OP mouse models, and induced osteoclasts. Inhibition of NEURL3 leads to a 20% decrease in BMM survival rate and a reduction in the number of tartrate-resistant acid phosphatase (TRAP) positive cells, which is a characteristic of mature osteoclasts. Furthermore, the expression levels of osteoclastogenesis-related marker genes were reduced by 50%. In vivo studies revealed that suppressing NEURL3 resulted in a 38% improvement in trabecular bone volume (BV/TV) and a 28% increase in bone mineral density (BMD) in the OVX-induced OP mice. Mechanistically, NEURL3 promoted osteoclast differentiation by increasing the ubiquitination levels of BMP7. Inhibition of BMP7 reversed the effects of NEURL3 on osteoclast differentiation in BMMs. Suppression of NEURL3 inhibits osteoclast differentiation of BMMs in vitro and alleviates bone loss in vivo. The underlying mechanism may involve NEURL3-induced ubiquitination of BMP7. Collectively, the downregulation of NEURL3 represents a promising therapeutic strategy for suppressing osteoclast differentiation and treating OP.