Warfarin Promotes Osteoclast Differentiation by Inhibiting the Expression of MGP through ERK Signaling

Abstract

Background: Osteoporosis, an osteolytic disease, is characterized by excess osteoclast activity. Warfarin, a widely prescribed oral anticoagulant, has been linked to an increased risk of long-term osteoporosis and fractures. However, the precise role of warfarin plays in osteoclast function remains unclear. Objective: This work aims to assess the impact of warfarin on the Receptor Activator of Nuclear Factor-κ B Ligand (RANKL)-caused osteoclastogenesis, as well as the underlying mechanisms. Methods: We extracted the bone marrow-derived macrophages (BMMs) from C57BL/6 mice (aged 6 weeks, male). A Microplate reader (450 nm, Tecan, RNE-90002, Hong Kong, China) was applied to assess the absorbance. Before the experiment, we extracted several drops of BMMs in equal quantities after cell culture for control and warfarin. The effect of warfarin on RANKL-modulated osteoclast differentiation was assessed by Tartrate-resistant acid phosphatase (TRAP) staining and F-actin detection, which was verified by examining osteoclast-related gene level. We also examined the expression of matrix gla protein (MGP) via Quantitative Real-time PCR (qRT-PCR) and western blot. Osteoclastogenesis-associated signaling pathways were assessed by western blot. Results: Warfarin significantly inhibits the expression of MGP (p < 0.05). TRAP staining illustrated that the warfarin treatment group formed more TRAP-positive osteoclasts. Under a fluorescence microscope, the area of F-actin rings was significantly increased in the warfarin treatment group (p < 0.01). On the other hand, Warfarin up-regulated osteoclast-related gene levels, including Cathepsin K (CTSK), TRAP, Calcitonin receptor (CTR), Dendritic cell-specific transmembrane proteins (DC-STAMP), Guided growth factor (c-Fos). Warfarin up-regulates the expression of RANKL-induced osteoclast differentiation marker genes (p < 0.01 or p < 0.001) significantly. Studies on molecular mechanisms have confirmed that warfarin inhibits the matrix gla protein (MGP) level and activates RANKL-induced ERK (extracellular signal-regulated kinase) signaling. Conclusions: These findings suggest that warfarins promotion of osteoclast differentiation may be linked to the inhibition of MGP and activation of ERK signaling. Consequently, this sheds light on the causes of bone loss associated with warfarin treatment and could have significant implications for clinical practice.